T Cell Hybridomas And Related Compositions And Methods For Assaying And Modulating T Cell Receptor-Mediated Immune Responses

ABSTRACT

The present invention provides T cell hybridomas and related compositions and assay systems for investigative, diagnostic, and therapeutic use in modulating T cell receptor (TCR)-mediated immune response. The T cell hybridomas of the invention are typically constructed by fusing a naïve or early central memory T cell isolated from a mammalian subject with an immortalizing fusion partner (e.g. mammalian lymphoid tumor cell) to yield clonal T cell hybrids. The resulting T cell hybridomas exhibit antigen (Ag)-specific proliferation responsiveness over a background level of proliferation of the hybridomas. These hybridomas are useful for screening, identifying, and characterizing T cell immune modulatory agents, for example recombinant T cell receptor ligands (RTLs) and other agents that can modulate TCR-mediated T cell immune responses. Ag-specific proliferative response profiles exhibited by the T cell hybridomas of the invention show sufficient amplitude, sensitivity and fidelity to distinguish and/or quantify the presence and/or activity of a test RTL or other test modulatory agent in screening and sensitivity assays employing the hybridomas. These and other aspects of the invention yield powerful tools and methods for developing and characterizing novel RTLs and other immune modulatory agents for use in treating immune disorders, including a wide range of autoimmune diseases, in mammals.

The invention relates to the field of immunology. More particularly, theinvention relates to hybridoma technology and related compositions andmethods for developing and evaluating diagnostic and therapeutic toolsfor modifying immune responses in mammalian subjects, including formanagement of autoimmune diseases, such as multiple sclerosis.

BACKGROUND OF THE INVENTION

Immune responses in mammals are mediated by a diverse collection ofperipheral blood cells called leukocytes. These cells arise fromhematopoietic stem cells, which undergo self-renewal and differentiationinto two precursor lineages—the myeloid and lymphoid lines. Furtherdifferentiation occurs among these lineages to produce monocyte,eosinophil, neutrophil, basophil, megakaryocyte, and erythroid cellsfrom the myeloid line, and T lymphocytes, B lymphocytes, and NK cellsfrom the lymphoid line.

T lymphocyte differentiation occurs in the thymus and proceeds throughprothymocyte, cortical thymocyte and medullary thymocyte intermediatestages, to produce various types of mature T cells. The principal Tlymphocyte subtypes include CD8⁺ T cells (also known ascytotoxic/suppressor T cells or CTLs), which, when activated, have thecapacity to lyse target cells, and CD4⁺ T cells (also known as T helperand T inducer cells), which, when activated, have the capacity toregulate other immune system cell types.

Immune system responses are elicited in several differing situations.The most frequent response is as a desirable protective response againstinfectious microorganisms. However, undesired immune responses can occurin the context of various autoimmune diseases, and followingtransplantation of foreign tissue. In the case autoimmune diseases, thebody's own antigens present targets for autoreactive immune responses.Immune responses can also be initiated in vitro by mitogens, antibodiesagainst certain receptors, and cognate antigens of T cells.

Immune responses are “transduced” from a stimulating event via a complexinteraction of leukocyte cell types and regulatory molecules. Theparticipating cell types and the nature of interactions between celltypes may vary for different stimulating events. For example, immuneresponses against invading bacteria are often transduced by formation ofcomplexes between an MHC Class II receptor and a bacterial antigen,which trigger activation of CD4⁺ T cells. By contrast, immune responsesagainst viral infections are principally transduced by formation of MHCClass I/viral antigen complexes and subsequent activation of CD8⁺ cells.

Activation of T lymphocytes occurs naturally when the T cells interactwith antigen-presenting cells (APCs) bearing cognate antigen (Ag) in thecontext of a major histocompatibility complex (MHC) protein. Thespecificity of T cell responses is conferred by a polymorphic,antigen-specific T cell receptor (TCR). In particular, most T cellactivation involves recognition by an α/β heterodimeric TCR of antigenthat has been processed and presented on the surface of APCs as peptidesbound to MHC molecules. TCR α and β chains are each derived frommultiple germline-encoded elements which undergo somatic recombinationduring T cell development. In the mouse, the repertoire of α chaingermline genes is thought to number approximately 100 different variable(V α) segments, and also 100 different joining (J α) segments. TCR βchains are derived from a repertoire of gene segments estimated toinclude some 25 different Vβ elements, 12 Jβ genes, and two diversity(Dβ)elements. Random joining of these various intra α and β chainelements, along with combinatorial associations of different α and βchains, permit a considerable degree of TCR diversity. In addition,during the somatic process of V/J recombination (V/D/J for β chains),further diversity is created in the junctional regions through theaddition of nongermline encoded nucleotides (N region). Given the numberof different germline elements, the number of possible junctional regionsequences, and the combinatory associations of the two chains, it hasbeen estimated that the potential TCR repertoire is of the order of1015-1022 specificities (Davis et al., Nature 334:395, 1988; Hunkapillaret al., Adv. Immunology 44:1, 1988)

In addition to the antigen specific TCR, a number of other cell surfaceproteins regulate T cell activation and impart sensitivity andflexibility to the immune response. These additional regulatory proteinsinclude the surface antigens CD2, CD4 CD8 and lymphocyte functionassociated antigen. Such surface antigens are non-polymorphic moleculesthat increase the avidity with which a T cell interacts with APCs ortarget cells, and also play a role in signal transduction.

CD4 and CD8 molecules are expressed on mutually exclusive populations ofmature T cells that bear TCRs specific for antigen in association withMHC class II, and MHC class I proteins, respectively. These moleculesenhance the avidity with which a T cell binds antigen-bearing or targetcells, and may also promote the interaction of the TCR with itsappropriate antigen. Bierer et al., Ann. Rev. Immunol. 7:579-99, 1989.

MHC-restricted T lymphocyte interactions have been widely andextensively investigated. Cells of the T helper/inducer subset generallyrecognize antigen on the surface of APCs only in association with classII MHC gene products, which results in genetic restriction of antigenrecognition. While the rules governing the activation of MHC-restrictedT cells, and particularly of class II MHC-restricted T cells, have beenwell described, the underlying mechanisms are still being defined.

Despite the very large number of possible TCR specificities of T cells,a number of studies have shown that the major portion of the T cellresponse to numerous different protein antigens may be directed to a few“immunodominant” epitopes within the antigenic protein. In the contextof autoimmune diseases, HLA-DR4-restricted T cell responses, and in somecases clinical signs of autoimmune disorder, have been demonstrated tobe associated with specific proteins and/or immunodominant epitopes fromthese proteins, including, e.g., type II collagen (Rosloneic et al., J.Immunol. 160:2573-78, 1998; Andersson et al., Proc. Natl. Acad. Sci. USA95:7574-79, 1998; and Fugger et al., Eur. J. Immunol. 26:928-33, 1996),and human cartilage Ag gp39 (Cope et al., Arthritis Rheum. 42:1497,1999) associated with rheumatoid arthritis (RA), glutamic aciddecarboxylase 65 (Patel et al., Proc. Natl. Acad. Sci. USA 94:8082-87,1997; Wicker et al., J. Clin. Invest. 98:2597, 1996) and insulin (Congiaet al., Proc. Natl. Acad. Sci. USA 95:3833-38, 1998) associated withType 1 diabetes (insulin dependent diabetes mellitus or IDDM), andmyelin oligodendrocyte glycoprotein (MOO) (Forsthuber et al., J.Immunol. 167:7119, 2001) associated with MS and an animal disease modelfor MS, experimental autoimmune encephalomyelitis (EAE). Similarfindings have been reported for HLA-DR2-restricted T cell responsesassociated with myelin basic protein (MBP) (Madsen et al., Nat. Genet.23:343, 1999), proteolipid protein (PLP) (Kawamura et al., J. Clin.Invest. 105:977, 2000), and MOG (Vandenbark et al., J. Immunol.171:127-33, 2003).

In addition to the phenomenon of immunodominant peptide recognition, TCRutilization in autoimmune responses is often quite limited, despite avast diversity of TCRs available in the T cell population. For example,limited TCR utilization has been reported in the immune response ofLewis rats immunized with myelin basic protein (MBP) to induce EAE. TheT cell response thus induced is directed primarily to an immunodominantepitope contained within an encephalitogenic fragment of MBP comprisingamino acids 66-88 of the protein (MBP 66-88). The T cell response isalso highly restricted, dominated by T cells expressing TCR Vβ8 almostexclusively and Vα2 frequently (Burns et al., J. Exp. Med. 169:27,1989). Similarly, Gold et al., J. Exp. Med. 174:1467-176, 1991 reportconserved TCR Vα and Vβ utilization in EAE, wherein the TCR β chainsequences of T cell clones and hybridomas reactive to MBP 68-88 allutilized a Vβ8.2 segment, and exhibited other conserved structuralfeatures.

One very promising approach for regulating antigen-specific T cellresponses in autoimmunity and in other contexts (e.g., graft rejection)is to reprogram or induce nonresponsiveness in T cells using recombinantor synthetic TCR ligands, or T cell modulatory drugs or other compoundsthat are agonists or antagonists for activation of TCRs by their cognateligands. In this regard, various analogs of TCR ligands have beenproduced which comprise extracellular domains of class II MHC moleculeslinked to specific peptide targets. Several such constructs have beendeveloped that involve natural or recombinant α1α2 and β1β2 MHC class IIdomains in association with various encephalitogenic or other pathogenicpeptides covalently linked or noncovalently bound to the MHC IIcomponent to form a complex (Kozono et al., Nature 369:151, 1994;Fremont et al., Science 272:1001, 1996; Sharma et al., Proc. Natl. Acad.Sci. USA 88:11405, 1991; Nicolle et al., J. Clin. Invest. 93:1361, 1994;Spack et al., CNS Drug Rev. 4: 225, 1998). These molecular complexesbind not only to the TCR but also to the CD4 molecule on the T cellsurface through the 132 MHC domain (Brogdon et al., J. Immunol.161:5472, 1998), and have been reported to inhibit T cell activation andprevent EAE in rodents (Sharma et al., Proc. Natl. Acad. Sci. USA88:11405, 1991; Spack et al., CNS Drub Rev. 4: 225, 1998; Steward etal., J. Allerg. Clin. Immun. 2:S117, 1997).

An even more promising design for TCR modulatory agents in this contextare recombinant T cell receptor ligands (RTLs) that incorporate aminimal TCR interface, for example comprising only the α1 and β1 MHCdomains (or otherwise excluding the β2 CD4-binding domain) covalentlylinked to peptide (Burrows et al., Prot Eng. 12:771, 1999). These RTLconstructs have been shown to prevent and treat MBP-induced EAE in Lewisrats (Burrows et al., J. Immunol. 161:5987, 1998; Burrows et al., J.Immunol. 164:6366, 2000) and to inhibit activation and induce IL-10secretion in human DR2-restricted T cell clones specific for MBP-85-95or BCR-ABL b3a2 peptide (CABL) (Burrows et al., J. Immunol. 167:4386,2001; Chang et al., J. Biol. Chem. 276:24170, 2001). Additional RTLconstructs have been designed and tested by inventors in the instantapplication, which include a MOG-35-55/DR2 construct (VG312) shown topotently inhibit autoimmune responses and lead to immunologicaltolerance to the encephalitogenic MOG-35-55 peptide and reverse clinicaland histological signs of EAE (Vandenbark et al., J. Immunol.171:127-33, 2003). Numerous additional RTL constructs that are usefulfor modulating T cell immune responses and can be employed within theinvention are described herein, below.

To evaluate the biological function and mechanisms of action of RTLs andother T cell modulatory agents, antigen-specific T cells bearing cognateTCRs have been used as target T cells for testing (see, e.g., Burrows etal., J. Immunol. 167:4386, 2001). However, a low frequency ofAg-specific T cells, varying levels of T cell Ag-specific responses, anda potential for uncontrolled interactions (e.g., with other, differentcells) have significantly limited the scope of these investigations.

Two basic strategies have been devised to isolate and propagatelymphocyte lines and clones of defined specificity for evaluating T cellfunction and modulation. One approach has been to clonally expand andpropagate normal immune lymphocytes through repetitive stimulation withantigen and/or growth factors. The second approach has been to produceimmortalized lymphocyte hybrids by somatic cell hybridization withcancer cells (e.g., lymphoma or myeloma cells).

The latter approach of “hybridoma” technology was discovered by Kohlerand Milstein (Nature 256:495, 1975), and was initially directed towardproduction of B lymphocyte hybrids (e.g., between B cells and immortal,plasmacytoma cells) to produce monoclonal antibodies. Subsequently,these methods were adapted toward production of functional, continuouslines of T lymphocytes.

Hybridomas are created from two cell populations by fusing the two celltypes together with a fusogen, such as polyethylene glycol (PEG). Theresulting fused cells (hybridomas) are isolated from unfused cells andused to establish continuous cell lines. Isolation of hybridomastypically involves the use of a selective medium, for example,hypoxanthine-aminopterin-thymidine (HAT) medium. The hybrid cells arethen expanded and subcloned to generate a pure cell line, ormonoculture.

Although the majority of hybridoma work to date has focused on B cellsfor producing monoclonal antibodies, the method has also beeneffectively used to make functional T cell hybrids. For example,Kappler, et al., (J. Exp. Med. 153:1198-1214, 1981) described murine Tcell hybridomas made by fusing an interleukin-2 (IL-2)-producing,HAT-sensitive murine T cell tumor line with normal murine T cell blastsenriched in H-2 antigen-specific cells. The resulting hybrids could beinduced to produce IL-2 by mitogen and/or antigens. Other early reportsof T cell hybridoma production are provided by Grillot-Courvalin, et al.(Nature 292:844, 1981), and Greene, et al. (Clin. Res. 29:368A, 1981).Grillot-Courvalin, et al. made a T cell hybridoma by hybridizing normalperipheral blood T cells with a HAT-sensitive T cell lymphoma. Greene etal. made a suppressor T cell hybrid by fusing peripheral bloodlymphocytes enriched for suppressor T cells (by treatment with themitogen, concanavalin A (ConA) with a HAT-sensitive T leukemia cellline.

Hybridoma methodology has more recently been employed for production ofantigen-specific, class II MHC-restricted, T-T hybridomas. Various classII MHC-restricted T-T hybridomas have been produced, and these hybridshave shown utility for various purposes, including the analysis of Tcell receptor structure-function, cell-cell interactions, and T cellactivation involving T helper cells (see, e.g., White et al., J.Immunol. 130:1033-37, 1983; Rock, K. L., “Functional T-cell Hybridoma”,in Hybridoma Technology in the Biosciences and Medicine, Ed. T. A.Springer, Plenum Press, N. Y., 1985; Allen et al., Proc. Natl. Acad.Sci. USA 81:2489-93, 1984; Allen et al., J. Exp. Med. 162:1264-68,1985a; Allen et al., J. Immunol. 135:368-72, 1985b; Marrack et al., Adv.Immunol. 38:1, 1986; Jarboe et al., Infect. Immun. 52:326-30, 1986;Bierer et al, Ann. Rev. Immunol. 7: 579, 1989; Peccoud et al., EMBO J.9:4215-23, 1990; Engleman et al., U.S. Pat. No. 4,950,598, issued Aug.21, 1990; U.S. Pat. No. 5,019,384, issued May 28, 1991, and Sypek etal., Infect. Immun. 58:1146-52, 1990).

A survey of more recent literature on T cell hybridoma research includesthe following reports:

White et al., (J. Immunol. 143:1822, 1989), and White et al. (J. Exp.Med. 177:119-25, 1993), generated hybridomas from a TCR-negative variantof the widely used AKR thymoma line BW5147. This BW5147 variant fusionpartner does not express functional TCR alpha- and beta-chains (asdetermined by surface TCR staining). These cells were fused with normalT lymphocytes for study at the clonal level (without interference offunctional, i.e., surface expressed, BW5147-derived TCR chains).

Gold et al., (J. Exp. Med. 174:1467-76, 1991) reported isolation of Tcell hybridomas produced by fusion of lymphoblasts from MBP-specific Tcell lines to a murine TCR α/β-(BW1100.129.237) cell line. The subjecthybridomas exhibited IL-2 production following stimulation with MBP(whole protein or synthetic peptide MBP 68-88) in the presence ofirradiated LEW spleen cells.

Michäelson et al., (Eur. J. Immunol. 22:1819-25, 1992) generated Thybridomas from rat CII-reactive T cell lines and myelin basic protein(MPB(89-101)-reactive T cells fused with the BW 5147 α-β-variant partneraccording to the method of White et al: (J Immunol. 143:1822, 1989). Thereactivity of these T cell hybridomas was again assayed indirectly,using an IL-2-dependent CTLL cell line. This activity was inhibited bypreincubation of spleen APCs with inhibitory peptides prior tostimulation of the hybridomas with stimulatory peptide.

Woods et al., (J. Exp. Med. 180:173-81, 1994) generated T cellhybridomas using T cells from a Tg murine subject in which the α1 and β1domains of mouse I-E^(d) were replaced by the corresponding domains ofhuman DRB1*0401 (Dr4Dw4) molecules (capable of presenting human classII-restricted peptides to mouse CD4-positive cells). The starting Tcells were isolated from lymph nodes of the Tg mice after immunizationwith a synthetic peptide from influenza virus hemaglutinin, HA(307-319),and were fused with the BW 5147 α-β-variant partner. The hybridomasresponded (as detected indirectly by IL-2 HT-2 bioassay) specifically toHA (107-319) presented by transgenic, but not nontransgenic, spleencells, and exhibited DR4Dw4 restriction specificity.

Liu et al., (J. Exp. Med. 186:1441-50, 1997), and Crawford et al.(Immunity 8:675-82, 1998) generated T cell hybridomas specific for awell-studied combination of MHC+peptide (M^(k)+moth cytochrome c 88-103(MCC)). Tg Mice were created that expressed genes coding for solubleIE^(k) covalently linked via a flexible linker at its NH2-tenninal endto peptides shown to bind intact IE^(k) protein (Kozono et al., Immunity3:187-196, 1995). Tg mice injected with IE^(k) bound to an MCC peptideproduced T cells that were fused with TCR α-/β-BW5147 variant thymomacells. The resulting hybridomas expressed high levels of TCR and CD4,and were assayed for their ability to be bound by and react with reactwith IE^(k) and different concentrations of MCC in the presence ofB10.BR spleen cells as APCs, or with a soluble, multimeric biotinylatedIE^(k)MCC ligand complex. Hybridoma responses were again measured byindirect IL-2 assay, which reportedly showed that the hybridomaresponses were CD4-dependent and IE^(k)-specific (as determined byanti-IE blockade).

Laufer et al., (J. Immunol. 162:5078-84, 1999) generated T hybridomasusing splenocytes derived from K14 mice (a Tg murine model forinflammatory skin disease) stimulated with irradiated B 6 splenocytesand fused with the BW 5147 α-β-variant fusion partner. B6-reactivehybrids were selected as positive if they showed a response overbackground in indirect IL-2 activation assays using the IL-2-dependentindicator cell line HT-2. (See, also, Fan et al., Proc. Natl. Acad. Sci.USA 100:3386-91, 2003).

Fukui et al., (Proc. Natl. Acad. Sci. USA 97:13760-65, 2000) made T cellhybridomas specific for pigeon cytochrome c-derived peptide, 50V,restricted by I-A^(b). The subject hybridomas were stimulated withirradiated LN cells and 50V peptide and assayed indirectly for IL-2production using CTLL.

Chen et al., (J. Virol. 74:7587-99, 2000) made hepatitis B virus (HBV) eantigen (HBeAg)-specific T cell hybridomas from T cells generated inHBeAg Tg (B10 e/e) mice and also using the BW 5147 α-β-variant fusionpartner. A panel of these hybridomas was used to identify animmunodominant (129-140/IA^(b)) epitope of HBeAg. TCR genes fromselected hybridomas were cloned and sequenced to elucidate V gene usage,and further data showed that multiple TCRs recognized the 129-140/IA^(b)epitope. Some of these hybridomas were selected as candidate donors ofTCR genes for generating TCR Tg mice.

Rosloneic et al., (J. Immunol. 160:2573-78, 1998) and Kotzin et al.,(Proc. Nat. Acad. Sci. USA 97:291-96, 2000) also made T cell hybridomasusing the BW5147 thymoma variant TCR α-/β-fusion partner. In the formerstudy, the authors examined the function of a RA susceptibility alleleHLA-DR4 (DRB1*0401) in presenting antigenic peptides derived from humantype II collagen (hCII). hCII specific lymphocytes were obtainedfollowing immunization of mice transgenic (Tg) for a chimeric HLA/I-EDR4 (in which the HLA second domains are exchanged for I-E, enablingmurine CD4 to interact with the chimeric molecule), and were fused withthe variant BW5147 thymoma cells. The resulting hybridomas were screenedfor their ability to recognize hCII presented by DR4 and I-A^(f)presented by syngeneic spleen cells. This assay similarly involvedindirect detection of hybridoma activation by IL-2 assay (measuring IL-2titers indirectly through the proliferative response of co-cultured HT-2cells).

In the Kotzin et al. study, hCII-specific T cell hybridomas were made byimmunizing DR4 Tg mice (DRB1*0401) with human CII, and fusing T cellsfrom the immunized Tg mice with the variant (TCR α-/β-) BW5147 thymomacells. Two hybridomas (DR4hCII-38.8 and DR4hCII-11.5) specific for thedominant CII determinant (CII 263-270) were generated and assayed fortheir ability to bind a labeled, soluble, tetrameric CII peptide-DR4complex. The CII DR4 tetramers bound in a specific manner to the T cellhybridomas that were previously shown (by indirect, IL-2 assay, asdescribed by Rosloneic et al., supra) to respond to the majorimmunodominant determinant, CII 263-270.

In this same study, Kotzin et al. made additional T cell hybridomastransfected to express chimeric TCR genes of clonal CD4+ expansionsidentified in RA synovial cells. The chimeric TCR genes were introducedinto TCR-negative and human CD4-expressing T cell hybridomas byelectroporation, and the transfectants were screened for surfaceexpression of TCR and shown to be functional by IL-2 release afterstimulation with a Vβ-selective superantigen. These transfectedhybridomas were control assayed for stimulation by CII(258-272) andHCgp39(263-275) peptides in the context of DR4 (*0401)-expressing APCs,and none reportedly responded.

Andersson et al., (Proc. Natl. Acad. Sci. USA 95:7574-79, 1998) used

HLA-DR (DRB1*0401, DRA1*0101), human CD4 Tg mice to generate Thybridomas responsive to an immunodominant T epitope in CII identifiedas CII 261-273, also using the variant TCR α-/β-BW5147 fusion partner.These hybridomas were incubated with a DRB1*0401-positive Epstein-Barrvirus-transformed B cell line (Priess), as APCs and varyingconcentrations of the CII peptide, and activation was determined by IL-2specific sandwich ELISA based on an Eu³⁺-labeled streptavidin detectionsystem (Fugger et al., Eur. J. Immunol. 26:928-33, 1996).

Backlund et al., (Proc. Nat. Acad. Sci. USA 99:9960-65, 2002) used ahumanized mouse model expressing HLA-DRB1*0401/DRA1*0101, human CD4, andhuman CII (huCII) on a background deficient of murine class IIexpression (Fugger et al., Proc. Natl. Acad. Sci. USA 91:6151 55, 1994;Malmström et al., Scand. J. Immunol. 45:670-77, 1997) to furtherelucidate the role and behavior of T cells in RA. From these studies theauthors reported a dominant T cell response to glycosylatedCII-glycopeptides in a cohort of severely affected RA-patients.

Maverakis et al., (Proc. Natl. Acad. Sci. USA 100:5342-47, 2003) made Thybridomas specific for an N-terminal autoantigenic peptide (Ac1-9) ofmyelin basic protein (MBP), again using the TCR α-/β-BW5147 variantfusion partner. Peptide-responsive hybridomas were also identified byindirect IL-2 production bioassay using HT-2 cells.

Strattman et al., (J. Clin. Invest. 112:902-14, 2003) reportedproduction of additional T cell hybridomas using T cells from NOD mice(an art accepted model for insulin-dependent diabetes mellitus, or IDDM)(see also, Haskins et al., Diabetes 37:1444, 1988). These hybrids weregenerated by fusing BW5147 cells with T cells isolated from NOD miceimmunized with a “2.5 mimotope” peptide with high agonistic activity forautoreactive BDC-2.5 T cells isolated from NOD mice and shown to bepathogenic in transfer experiments (Judkowski et al., J. Immunol.166:908-17, 2001; Pankewycz et al., Eur. J. Immunol. 21:873-79, 1991;Daniel et al., Eur. J. Immunol. 25:1056-62, 1995); Haskins et al.,Science 249:1433-36, 1990). These hybridomas were specifically bound bythe 2.5 mimotope peptide complexed to A^(g7) to form an MHC-peptidetetramer complex (A^(g7) is the sole MHC class II molecule of NOD mice).As in previous studies, activation of these hybridomas in response toantigen (purified from pancreatic islets and presented by NODsplenocytes as APCs) was determined indirectly by IL-2 assays measuringproliferation of an IL-2 dependent cell line (NK) (Strattman et al.,supra).

In a separate IDDM-related study by Patel et al., (Proc. Natl. Acad.Sci. USA 94:8082-87, 1997), the BW5147 α-/β-variant was used as a fusionpartner with T cells isolated from DR0401, hCD4, I-AB (C-line) miceimmunized with purified recombinant human glutamic acid decarboxylase 65(GAD65) protein. Over 200 hybridomas thus produced were reported to bespecific for GAD65 by using spleenic APCs from DR0401, hCD4, I-Aβ mice.The GAD-65-specific hybridomas were tested against pools of overlappingGAD65 peptides in the presence of APCs (Tg mouse spleen cells, orEpstein-Barr virus (EBV)-transformed lymphoblastoid cells), andhybridoma activation was assayed by an IL-2 sandwich immunoassayemploying a streptavidin-europium detection system. This epitope mappingstudy revealed an immunodominant core motif of GAD65 (LYNIIKNREG), whichincluded a DR0401 motif (YNIIKNREG) and a DR0405 motif (LYNIMNRE)(Rammensee et al., Immunogenetics 41:178-228, 1995). For each of 10identified epitopes, the authors reported verified MHC restriction byantibody blocking experiments (anti-DR antibodies reportedly blockedhybridoma activation, whereas an isotype-matched control antibody didnot affect the antigen-specific response).

In a related study, Congia et al., (Proc. Natl. Acad. Sci. USA95:3833-38, 1998) fused T cells from DRB1*0401, hCD4+, I-Aβ^(0/0) miceimmunized with purified recombinant human proinsulin or preproinsulinwith TCR α-/β-variant BW5147 cells. A panel of hybridomas was initiallyscreened using whole protein (insulin, proinsulin, or preproinsulin) andlater using overlapping peptides, and hybridoma activation wasindirectly measured by a europium-based IL-2 immunoassay. Using thispanel of hybridomas, an immunodominant epitope (pgs. 73-90) wasidentified spanning the C peptide and the A chain, another epitope (pgs.11-26) in the leader sequence, another spanning the leader and b chain(pgs. 20-36), and a fourth specific for the A chain (pgs. 85-101),indicating that the major T cell determinant of insulin and itsprohormones is located in the precursor forms of the protein.

Yang et al., (Proc. Natl. Acad. Sci. USA 99:6204-6209, 2002) constructedan alternative “THZ” T cell hybridoma by fusing activated mouse CD4+ Tcells with the BWZ cell line containing a reporter gene (lacZ) expressedunder control of an element of the IL-2 promoter (nuclear factor ofactivated T cells (NFAT)). A single THZ clone was isolated that lackedTCR expression but maintained CD3 and CD4 expression. This hybridomareportedly allows for detection of surface expression and specificity ofTCRs on the hybridomas by assaying lacZ activation in response toantigen.

Recently, Wang et al. (J. Immunol. 171:1934-40, 2003) used a rat T cellhybridoma specific for guinea pig (GP)-MBP (originally developed by Goldet al., J. Exp. Med. 174:1467-76, 1991) as a clonal target to examinethe function of RTLs. In this study, Wang and colleagues demonstratedthat the T cell hybridoma was selectively, but only partially, activatedto transduce early signaling through the TCR in an Ag-specific fashion.

The foregoing T cell hybridomas have essentially limitless growthpotential and grow readily under standard culture conditions.Furthermore, the immunological activity of these hybridomas is notsubject to cyclic fluctuations, as is seen with restimulated normalclones. T-inducer hybridomas produce lymphokines in response to T cellreceptor (TCR) stimulation, which provides a useful assay for examiningT cell activation events. Moreover, since T-inducer hybrids do notgenerally require costimulatory signals, it has been possible tostimulate them with fixed or disrupted antigen presenting cells as wellas model membrane systems (see, e.g., Shimonkevitz, et al., J. Exp. Med.158:303, 1983; Watts, et al., Proc. Natl. Acad. Sci. USA 81:1883, 1984).Therefore, antigen-specific class II MHC restricted T cell hybridomashave provided improved tools for analyzing the events in T cell Agpresentation and TCR-mediated regulation of T cell biology.

With respect to class I MHC-restricted hybridomas, these have also beenmade using immortalizing fusion partners. Whitaker et al., (J. Immunol.129:900-03, for example, described the preparation of H-2 restricted,reovirus-specific cytotoxic T cell hybrids using BW5147 as a fusionpartner. These hybrids, however, require a mitogenic lectin forstimulation and do not respond well to antigen and appropriate APCsalone. Other examples of class I MHC-restricted hybridomas generatedwith BW5147 cells have been reported by Kaufmann et al., (Proc. Natl.Acad. Sci. USA 78:2502, 1981), and by Endres et al., (J. Immunol.131:1656, 1983). However, such hybrids are identified at low frequencyand may represent “high affinity” T cells that are not CD8 dependent(see, e.g., MacDonald et al, Immunol. Rev. 68:89, 1982). Furthermore,the successful isolation of such hybridomas has been limited to thosethat are MHC reactive (e.g., allo- or auto-reactive) and has not beendescribed for antigen-specific, MHC-restricted cells.

Despite the wide ranging and long enduring efforts evinced by theforegoing reports, the goals of developing useful, clonal T cell linesfor analyzing T cell function and identifying and characterizing T cellmodulatory agents, remain only partially fulfilled. The utility ofexisting T cell hybridomas for these purposes remains seriously limited.A principal drawback of current technologies in this regard relates tothe inherently high proliferation potential of immortalizing T cellfusion partners for generating T hybridomas (as driven constitutively,for example, by oncogen promoters). Because of this high intrinsicproliferative capacity exhibited by the requisite fusion partner, assaysto detect and characterize Ag-specific, TCR-mediated activationresponses by T cell hybridomas have almost universally focused on asingle indicator of T cell activation, IL-2 production. Regardless ofthe IL-2 assay format used, the resulting data are necessarily limitedin terms of detecting, characterizing, and/or measuring meaningful Tcell regulatory events. The same inherent limitation applies to otherknown T hybrid activation assays, for example as described by Yang etal., (Proc. Natl. Acad. Sci. USA 99:6204-09, 2002). In the Yang et al.study, “THZ” hybridomas have been characterized by a limited Ag-specificactivation profile—characterized by lacZ reporter expression which isalso linked to a discrete activation event of IL-2 gene activation.

A more comprehensive biological assay for T cell activation, indicativeof a broader assemblage of T cell activation mechanisms and events, istherefore highly desired. A widely recognized T cell response in thiscontext is represented by Ag-specific T cell proliferation, whichresponse indicates a much broader and biologically significantactivation profile than a single cytokine upregulation event. However,as noted above, previously developed T cell hybridomas have not beenshown to exhibit useful, Ag-specific proliferative responses in thiscontext, and available assays have focused almost exclusively onindirect measurements of IL-2-induced proliferation of cocultured cells.This deficiency may be attributed, at least in part, to the highbackground proliferation imparted to T cell hybridomas by theimmortalizing fusion partner, which are required for generating clonalhybrids.

By virtue of their high constitutive or baseline proliferation capacity,existing T cell hybrids fail to exhibit meaningful, Ag-specificproliferation responses over background. More importantly, the maskingbaseline proliferative potential of known T cell hybridomas makes theseclones refractory to studies of RTLs and other T cell modulatory agents.In particular, these hybrids are not amenable to assays for detectingand/or measuring agonistic or antagonistic effects of RTLs and other TCRmodulatory agents (i.e., in comparison to a “control” proliferativeresponse elicited and observed in the hybrids following cognateAg-stimulation in the absence of the RTL or other modulatory agent).Even if such a control or baseline proliferative response abovebackground could be discerned in a known T cell hybridoma afterAg-stimulation, the subject hybridoma would not exhibit the necessaryfidelity and sensitivity of an Ag-specific proliferative response topermit detection and/or quantification of a more attenuated,anti-proliferative or pro-proliferative effect (e.g., as would have tobe discerned following pre- or post-incubation of Ag-stimulated hybridswith a test RTL or other T cell modulatory agent, in comparison to suchcontrol or baseline proliferative response in the absence of the testagent).

Accordingly, there remains a compelling need in the art for T cellhybridomas that exhibit a readily-detectable and quantifiable,Ag-specific proliferation response over a background level ofproliferation of the hybridomas (inclusive of a resting or constitutiveproliferation rate, and of a non-specific activation proliferation levelafter stimulation by mitogen and/or APCs in the absence of cognateantigen).

A related need exists for T cell hybridomas that exhibit an Ag-specificproliferation response profile suitable for detecting and/or measuringagonistic and antagonistic effects of RTLs and other TCR modulatoryagents on Ag-induced proliferation of the hybridomas.

It is therefore an object of the invention to provide T cell hybridomasthat exhibit a biologically meaningful Ag-specific proliferationresponse over a background level of proliferation of the hybridomas. Itis a further object of the invention to provide T cell hybridomas thatexhibit an Ag-specific proliferative response following cognateAg-stimulation in the absence of a test RTL or other test modulatoryagent, that is of sufficient sensitivity and fidelity to distinguishand/or quantify the presence and/or activity of the test RTL or othertest modulatory agent in a screening or sensitivity assay culture of thehybridomas.

Yet another object of the present invention is to provide quantitativeand sensitive assays for the identification and characterization of Tcell modulatory RTLs and other T cell modulatory agents useful formodulating TCR-mediated, T cell immune responses in investigative,diagnostic, and/or therapeutic applications.

The present invention fulfills these objects and satisfies additionalobjects and advantages, as will become apparent from the followingdescription.

SUMMARY OF EXEMPLARY EMBODIMENTS

The present invention provides T cell hybrids and related assay systemsthat allow for the screening, design, construction, and characterizationof novel immune modulatory agents, including recombinant T cell ligands(RTLs).

The T cell hybrids of the invention are typically produced by fusing amammalian T cell that expresses a T cell receptor (TCR) with a mammalianfusion partner cell to yield a clonal hybrid. The T cell hybrids exhibitan antigen (Ag)-specific, TCR-mediated proliferative response whenstimulated by cognate Ag. This Ag-specific proliferative response can bedetected above a background or resting proliferation rate of the T cellhybrids.

The T cell hybrids of the invention also display an Ag-specific,TCR-mediated proliferative response following contact with cognate,which can be detectably inhibited or stimulated by contacting the T cellhybrid with a TCR antagonist or TCR agonist. The Ag-specific,TCR-mediated proliferation kinetics of the T cell hybrids permit use ofthe T cell hybrids in screening and quantitative assays to detect thepresence, concentration and/or activity of TCR antagonists and agonists.

The invention thus provides a sensitive biological assay forTCR-mediated T cell activation, allowing for reliable detection of bothAg-stimulated and agonist- or antagonist-mediated effects on TCRfunction.

The methods and compositions of the invention provideproliferation-based assays that indicate a broader, more biologicallysignificant range of TCR-activation events. These methods andcompositions are therefore readily incorporated in assays to screen,identify, and characterize immune modulatory agents that can alterTCR-mediated T cell immune responses. Within these novel assay tools andmethods, the T cell hybrids of the invention exhibit modified,Ag-specific, TCR-mediated proliferation responses that accurately andreproducibly indicate a presence, quantity, and/or activity level of aselected RTL or other TCR modulatory agents in contact with the T cellhybrid. In related aspects of the invention, the T cell hybrids areemployed in assays for detecting and/or measuring the presence,concentration and/or activity of a T cell agonist or T cell antagonistthat may be added as a test agent to a culture of the T cell hybrids andobserved for their modulatory effects on TCR-mediated activation ofhybrid proliferative and other activation responses. In other aspects ofthe invention, the T cell hybrids are employed in a range ofinvestigative, diagnostic, and clinical applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. T cell lines specific for mouse (m)MOG-35-55 and human(h)MOG-35-55 peptides from splenocytes of HLA-DRB1*1501-Tg mice. A)Splenocytes from mMOG-35-55 peptide-immunized DRB1*1501-Tg mice at2^(nd) stimulation with 0.08 to 50 μg/ml mMOG-35-55 peptide; B)Splenocytes from hMOG-35-55 peptide-immunized 1501-Tg mice at 2^(nd)stimulation with 0.08 to 50 μg/ml hMOG-35-55 peptide; C) A T cell lineselected from a DRB1*1501-Tg mouse immunized with mMOG-35-55 peptide at2^(nd) stimulation reactive to mMOG-35-55 and cross-reactive withhMOG-35-55 peptides in a reciprocal dose-dependent fashion.

FIG. 2. Expression of TCR V gene segments in BW5147, BW5147 variant andT cell hybrids. TCR BV and AV gene expression was detected by RT-PCR.Top panel: BW5147.G.1.4 thymoma cell line purchased from ATCC (#TIB 48).Second panel: H6-1 hyrid clone, a T cell from mMOG-35-55peptide-immunized DRB1*1501-Tg mice fused with a BW5147 cell. Thishybrid was specific for mMOG-35-55 peptide (SI=4×) and cross-reactivewith hMOG-35-55 peptide (SI=4×). Third panel: BW5147 variant cell line.Fourth panel: H2-1 hybrid clone, a T cell from hMOG-35-55peptide-immunized DRB1*1501-Tg mice fused with a BW5147 variant cell.This hybrid was specific for hMOG-35-55 peptide (SI=12×) andcross-reactive with mMOG-35-55 peptide (SI=10×). Nomenclature:A10=AV19S1; BM.A=AV18S2; BM.B=AV17S3; BW.B=AV16S1P.

FIG. 3. Surface and intracellular staining of anti-TCR beta andanti-AV11 on BW5147, BW5147 variant and T cell hybrids. A) 0.5×10⁶ cellsof BW5147, BW5147 variant, H6-1 and H2-1 were stained with anti-TCRbeta-Cy-Chrome or anti-mouse IgG 2a-Cy-Chrome. B) 1×10⁶ cells from eachof the above samples were incubated with Brefeldin A for 6 hr followedby surface staining with anti-CD4-FITC, fixation, and permeabilization.Permeabilized cells were further stained with anti-TCR beta-Cy-Chrome.C) 1×10⁶ cells from H2-1, H6-1 and BW5147 were stained withanti-AV11-FITC for 45 min followed by FACScan analysis.

FIG. 4. Phenotyping of MOG-35-55 peptide-specific T cell hybrids. A)1×10⁶ cells of H2-1, H6-1 and BW5147 were stained with anti-CD3-FITCalone, or anti-CD4-PE plus anti-CD8-FITC, or anti-mouse IgG1-PE. B)Intracellular expression of IL-2, TNF-α and IFN-γ by ConA-activated Tcell hybrid H2-1.

FIG. 5. Ag-specific responses by T cell hybrids. A) H2-1 selected from384 isolates of H2 hybrid was tested for its response to hMOG-35-55peptide in both a direct proliferation in the presence ofDRB1*1501-transfected L cells and an indirect assay using 50%supernatant on CTLL-2 cells for 24 hr. B) H6 and A5 hybrids were derivedfrom mMOG-35-55 peptide-specific T cells and tested for responses tomMOG-35-55 peptide and ConA in an indirect assay. C) H2 hybrid wastested for cross-reactivity to mMOG-35-55 peptide and both H6 and A5hybrids were tested for cross-reactivity to hMOG-35-55 peptide in thepresence of DRB1*1501-transfected A PC. 50% supernatants were assayed.D) H2 hybrid was cultured with syngeneic APC in the presence of 10 μg/mlhMOG-35-55, MBP-85-99, or PLP-95-116 peptides for 72 hr and 50%supernatants were assayed. Student's t test was used. E) H2 hybrid cells(40,000 per well in triplicate) were added with either 1×10⁵ irradiatedsyngeneic APC or 20,000 irradiated (4500 rad) transfected L cellsexpressing DRB1 *1501 or DRB5*0101 in the presence of 10 μg/ml eitherMOG-35-55 peptides or 5 μg/ml ConA for 72 hr, and the supernatants wereassayed on CTLL-2 cells for 24 hr.

FIG. 6. Biological functions of hMOG-35-55-specific RTL detected by H2-1hybrid in vitro and verified in hMOG-35-55-induced EAE. A) 2×10⁵/wellH2-1 hybrid cells in triplicate were cultured in 2 mM Tris-containingmedium alone, or with 8 uM RTL VG1000 or 8 uM RTL340 (specific forMBP-85-99) for 24, 48, 72 and 96 hr. 0.5 uCi [³H]Thymidine wasincorporated for the last 6 hr of each culturing time. Proliferation ofcultured H2-1 hybrid cells was compared among three different cultureconditions at each culturing time. Asterisks indicate significantlyreduced responses (p<0.05, Student's t test) between RTL-treated vs.buffer treated hybrid cells. B) Aliquotted H2-1 hybrid cells or Alhybrid cells were collected after 24 and 96 hr co-culture with 8 uM RTLVG1000 and washed three x with RPMI and re-plated in a new culture platewith 1×10⁵ hybrid cells per well, plus irradiated DRB1*1501-transfectantAPC with and without 10 ug/ml hMOG-35-55 peptide (for H2-1), orirradiated rat thymocytes with and without 10 ug/ml GP-BP-69-89 peptide(for A1). After 48 hr culture, 100 ul supernatant was collected fromeach well and transferred correspondingly into a new plate in which 5000CTLL-2 cells suspended in 100 ul per well were already placed. 0.5 uCi[³H]Thymidine was incorporated for the last 6 hr of a 24 hr culture.Proliferation of CTLL cells was measured by net cpm over background cpmthat was less than 1000-1500. C) Nine DRB1*1502-Tg male and female micewere immunized s.c. with hMOG-35-55 peptide in CFA. In addition, micewere given pertussis toxin (Ptx) on days 0 and 2 postimmunization. Afteronset of clinical signs, three mice were treated i.v. with 100 ul of 1mg/ml (40 uM) RTL VG1000 daily for 8 days and the other 6 mice wereinjected i.v. with 100 ul of 10 mM Tris buffer daily for 8 days. Theaverage daily EAE score was determined for each group of mice by summingthe individual scores and dividing by the number of mice in the group.

DESCRIPTION OF EXEMPLARY MODES OF INVENTION

The present invention provides novel mammalian T cell hybrids that areuseful in a variety of methods and compositions. In one embodiment ofthe invention, the T cell hybrids described herein are used in screeningand other assay compositions and methods to screen, identify, developand/or characterize immune modulatory agents that regulate T cellfunction. In related embodiments, the T cell hybrids are employed inassays for detecting and/or measuring the presence, concentration and/oractivity of T cell agonists or T cell antagonists that modulate T cellreceptor (TCR)-mediated, T cell immune responses. Within exemplaryembodiments, the T cell hybrids are used for identifying and/orcharacterizing recombinant T cell receptor ligands (RTLs) that regulateT cell function through the TCR. In other aspects of the invention, theT cell hybrids are employed in a range of investigative, diagnostic, andclinical applications.

The T cell hybrids of the invention are typically produced by fusing amammalian T cell that expresses a TCR with a mammalian fusion partnercell to yield a clonal T cell hybrid. Surprisingly, the clonal T cellhybrids of the invention exhibit an antigen (Ag)-specific, TCR-mediatedproliferative response when the hybrids are stimulated by cognate Ag.This proliferative response may occur upon stimulation by Ag alone,and/or following stimulation by Ag in the presence of a suitable,typically allogeneic, Ag-presenting cell (APC). This Ag-specificproliferative response can be detected above a background or restingproliferation rate of the T cell hybrids. For example, the T cellhybrids of the invention will exhibit a detectable increase inproliferation in response to stimulation by cognate Ag and/or APC, whichcan be accurately distinguished from a proliferation rate of the Thybrids without Ag stimulation, and/or following non-specificstimulation (e.g., by a mitogen, such as concanavalin A (ConA), or by acytokine, such as IL-2).

In more specific embodiments, the T cell hybrids of the inventiondisplay an Ag-specific, TCR-mediated proliferative response followingcontact with their cognate Ag (Ag alone, or Ag combined with APC), whichcan be detectably inhibited or stimulated by contacting the T cellhybrid with a TCR antagonist or TCR agonist. According to this aspect ofthe invention, the Ag-specific, TCR-mediated proliferation kinetics ofthe T cell hybrids exhibit sufficient fidelity and reproducibility topermit use of the T cell hybrids in screening and quantitative assays todetect the presence, concentration and/or activity of TCR antagonistsand agonists. When the T cell hybrids are contacted with a selected TCRagonist or antagonist, optionally before or after Ag-stimulation, amodified, Ag-specific, TCR-mediated proliferation response of the hybridcells can be observed. Detection or measurement of this modifiedproliferation response indicates a presence, quantity, and/or activitylevel of the T cell antagonist or T cell agonist in contact with the Tcell hybrid. In exemplary embodiments, the T cell hybrids exhibit anAg-specific, TCR-mediated proliferative response following contact withthe cognate Ag and APC that can be detectably inhibited or stimulated bycontacting the T cell hybrid with a selected RTL. This aspect of theinvention yields sensitive, quantitative methods and compositions usefulfor designing, screening, selecting, developing, and characterizingnovel or modified RTLs.

The invention thus provides a sensitive and comprehensive biologicalassay for TCR-mediated T cell activation. Because the T cell hybridsexhibit such unexpected amplitude and fidelity of proliferativeresponsiveness relative to background, they allow for reliable detectionof both Ag-stimulated and agonist- or antagonist-mediated effects on TCRfunction. This permits broad-mechanistic identification andcharacterization of TCR-mediated T cell activation. The related Tcell/TCR activation assays of the invention are therefore significantlymore meaningful and useful than assays based on previously-described Tcell hybrids—which have not been shown to exhibit useful, Ag-specificproliferative responses or agonist/antagonist effects on Ag-stimulatedproliferation (due, at least in part, to the masking influences of theirhigh constitutive or baseline proliferation capacity). Whereas prior Tcell hybrid-based assays focused necessarily on limited, often indirectactivation indices (e.g., indirect measurement of interleukin (IL)-2production), the methods and compositions of the invention provideproliferation-based assays that indicate a broader, more biologicallysignificant range of TCR-activation events. The methods and compositionsof the invention are therefore uniquely amenable for screening,identifying, and characterizing RTLs and other TCR modulatory agentsuseful for modulating T cell activity in vivo, for example to prevent ortreat autoimmune diseases. Within these novel assay tools and methods,the T cell hybrids of the invention exhibit modified, Ag-specific,TCR-mediated proliferation responses that accurately and reproduciblyindicate a presence, quantity, and/or activity level of a selected RTLor other TCR modulatory agents in contact with the T cell hybrid.

The T cell hybrids of the invention typically display a proliferationresponse to their cognate Ag in the presence of APCs that is two-fold orgreater than a control or baseline level of proliferation of thehybrids. This proliferation response may be measured in comparison to abaseline proliferation level of the T cell hybrids under similar cultureconditions but in the absence of cognate Ag and APCs. Alternatively, theAg-specific proliferation response may be exhibited over a non-specificcontrol activation level induced, for example, by cytokines (e.g.,IL-2), APCs alone, APCs in combination with a different, non-specificantigen, non-specific antigen alone, and/or mitogen (e.g., ConA). Incertain embodiments, the T cell hybrids of the invention exhibit a3-fold or greater proliferative response to cognate Ag/APC stimulationabove baseline or above proliferation levels of non-specific activationcontrols. In other embodiments, the Ag/APC proliferative response is5-fold or greater than baseline or non-specifically activated T hybridproliferation levels. In certain embodiments the Ag/APC proliferativeresponse is 8-fold or greater, 10-fold or greater, and up to 15-fold orgreater compared to baseline or non-specifically activated T hybridproliferation levels. These amplitudes, and the fidelity andreproducibility, of the Ag-specific proliferation profiles of the Thybrids of the invention provide for effective employment of the Thybrids in sensitive, quantitative assays and related compositionsuseful for designing, screening, selecting, developing, andcharacterizing novel or modified RTLs and other TCR modulatory agents.

For use within the methods and compositions of the invention, antigenpresenting cells (APCs) include dendritic cells, macrophages, B cells,and other cells that can process and present antigenic peptides inassociation with class I or class II MHC molecules and deliver aco-stimulatory signal necessary for T cell activation.

The T cell hybrids of the invention typically express CD3 and CD4, andoften display a Thl cytokine expression profile (see, e.g., Kerlero, J.Autoimmunity 11:287-99, 1998). In certain embodiments, the T cellhybrids express detectable levels of IL-2, tumor necrosis factor(TNF)-α, and interferon (IFN)-γ, either constitutively or in response tonon-specific (e.g., ConA) stimulation and/or Ag-specific stimulation.Commonly, the T cell hybrids exhibit significantly enhanced (e.g.,20-30%, 30-45%, or greater increased levels) of IL-2, TNF-α, TNF-β,and/or IFN-γ expression in response to non-specific stimulation and/orAg-specific stimulation. In more detailed embodiments, the T hybridcells may express low or undetectable levels of IFN-γ, consitutively,and the levels of IFN-γ expression may be lower constitutively orfollowing stimulation than levels ekhibited by T effector memory (Tem)cells (see below). In related embodiments, the T hybrid cells expresslow or undetectable levels of Th2 cytokines, including IL-4, IL-5 and/orIL-10. Other Th2 cytokines not typically expressed at substantial levels(i.e., at comparable levels to other known Th2 cells) by T cell hybridsof the invention include, but are not limited to, IL-6 and IL-13.Frequently, Th2 cytokine expression is not significantly elevated, andis more commonly low or undetectable, following non-specific stimulation(e.g., following exposure of the cells to ConA).

In additional embodiments of the invention, the T cell that is used toform the T cell hybrid is selected from naïve T cells or central memoryT cells (Tcms). Typically, the starting T cells are obtained from a bonemarrow, spleen, or draining lymph node of a mammalian subject followingimmunization of the subject with the cognate Ag.

Identification and characterization of naïve and Tcm starting T cellsfollows generally the teachings of Gudmundsdottir et al., J. Immunol.162:5212-23, 1999; Sallusto et al., Nature 401:708-712, 1999; Gramagliaet al., J. Immunol. 162:1333-1338, 1999; Campbell et al., J. Immunol.166:877-84, 2001, Younes et al., J. Exp. Med. 198:1909-22, 2003, Geginatet al., Pathol. Biol. 51:64-66, 2003, and Morel et al., Eur. J. Immunol.33:3212-19, 2003. The use of nave and/or Tcm starting T cells stronglycontributes to the amplitude and fidelity of the Ag-specificproliferation response profile of the resulting T hybrid cells.

Certain starting T cells for use in constructing the T cell hybrids ofthe invention are selected for positive expression of CD45. Typically,CD45 expression is high in naïve T cells, and low or undetectable in Tcmand T effector memory (Tem) cells. Within these embodiments, thestarting T cell may be a non-human mammalian T cell that is CD45RB+.Alternatively, the starting T cell may be a human T cell that isCD45RA+. In either case, a CD45+ phenotype will often be marked by CD45expression at levels equal to, or slightly to moderately diminishedfrom, levels of CD45 expression characteristic of naïve T cells (i.e.,obtained from the same subject or species), and substantially higherthan CD45 expression levels found in Tcm or Tem T cells. CD45+ T cellsfor use within the invention include activated naïve T cells which maybe harvested from primary lymphoid organs of animals immunized withcognate Ag. Activated naïve T cells most commonly used as starting Tcells express a CD45+ phenotype as described above, but may otherwisedepart phenotypically from other nave T cells in expression of one ormore other markers indicative of naïve T cell development, activation,and/or differentiation.

Within other aspects of the invention, the starting T cells are selectedfor positive expression of CD62L. Typically, CD62L expression is high innaïve T cells, and low or undetectable in Tem cells. Within theseembodiments, CD62L expression will be often be exhibited at levels equalto, or slightly (e.g., 10-20%) to moderately (20-45%) diminished from,levels of CD62L expression characteristic of naïve T cells and/or Tcmcells, and substantially greater than (e.g., increased by 50% or morecompared to) CD62L expression levels found in Tem cells. CD62L+ T cellsfor use within the invention include activated naïve T cells which maybe harvested from primary lymphoid organs of animals immunized withcognate Ag. CD62L+ T cells most commonly used as starting T cellsexpress a CD62L+ phenotype as described above, but may otherwise departphenotypically from naïve T cells and or Tcm cells in expression of oneor more other markers indicative of naïve T cell and/or Tem celldevelopment, activation, and/or differentiation.

Within other aspects of the invention, the starting T cells are selectedfor positive expression of CD27. Typically, CD27 expression is high innaïve T cells and Tem cells, and low or undetectable in Tem cells.Within these embodiments, a CD27+ phenotype is marked by CD27 expressionat levels equal to, or slightly (e.g., 10-20%) to moderately (20-45%)diminished from, levels of CD27 expression characteristic of naïve Tcells and/or Tem cells, and substantially greater than (e.g., increasedby 50% or more compared to) CD27 expression levels found in Tem cells.CD27+ T cells for use within the invention include activated naïve Tcells and Tem cells which may be harvested from primary lymphoid organsof animals immunized with cognate Ag. Activated naïve T cells and Temcells most commonly used as starting T cells express a CD27+phenotype asdescribed above, but may otherwise depart phenotypically from naïve Tcells and/or Tem cells in expression of one or more other markersindicative of naïve T cell and/or Tem cell development, activation,and/or differentiation.

Within other aspects of the invention, the starting T cells are selectedfor positive expression of CCR7. Typically, CCR7 expression is high innaïve T cells and Tem cells, and low or undetectable in Tem cells.Within these embodiments, a CCR7+ phenotype is marked by CCR7 expressionat levels equal to, or slightly (e.g., 10-20%) to moderately (20-45%)diminished from, levels of CCR7 expression characteristic of naïve Tcells and/or Tem cells, and substantially greater than (e.g., increasedby 50% or more compared to) CCR7 expression levels found in Tem cells.CCR7+ T cells for use within the invention include activated naïve Tcells and Tcm cells which may be harvested from primary lymphoid organsof animals immunized with cognate Ag. Activated naïve T cells and Tcmcells most commonly used as starting T cells express a CCR7+ phenotypeas described above, but may otherwise depart phenotypically from naïve Tcells and/or Tcm cells in expression of one or more other markersindicative of naïve T cell and/or Tcm cell development, activation,and/or differentiation.

Within other aspects of the invention, the starting T cells are selectedfor positive expression of lymphotoxin αβ (LT αβ). Typically, LT αβexpression is detectable 24-72 hours after activation of naïve T cells,and declines thereafter to become relatively low in Tem cells (see,e.g., Gudmundsdottir et al., J. Immunol. 162:5212-23, 1999). Withinthese embodiments, a LT αβ+ phenotype is marked by LT αβ expression atlevels equal to, or slightly (e.g., 10-20%) to moderately (20-45%)diminished from, levels of LT αβ expression characteristic of activatednaïve T cells and/or Tem cells, and substantially greater than (e.g.,increased by 50% or more compared to) LT αβ expression levels found inTem cells. LT αβ+ T cells for use within the invention include activatednaïve T cells and Tem cells which may be harvested from primary lymphoidorgans of animals immunized with cognate Ag. Activated naïve T cells andTem cells most commonly used as starting T cells express a LT αβ+phenotype as described above, but may otherwise depart phenotypicallyfrom naïve T cells and/or Tem cells in expression of one or more othermarkers indicative of naïve T cell and/or Tem cell development,activation, and/or differentiation.

Within other aspects of the invention, the starting T cells are selectedfor negative or low expression of the T cell differentiation markerCD44. Typically, CD44 expression is low or undetectable in naïve Tcells, and comparably high in Tem cells. Within these embodiments, aCD44-phenotype will be marked by CD44 expression at levels equal to, orslightly (e.g., 10-20%) to moderately (20-45%) increased from, levels ofCD44 expression characteristic of naïve T cells, and substantially lowerthan (e.g., less than 50% compared to) CD44 expression levels found inTem cells. These CD44− T cells for use within the invention may beharvested from primary lymphoid organs of animals immunized with cognateAg. Activated naïve, CD44− T cells most commonly used as starting Tcells express a CD44− phenotype as described above, but may otherwisedepart phenotypically from naïve T cells and/or Tcm cells in expressionof one or more other markers indicative of naïve T cell and/or Tcm celldevelopment, activation, and/or differentiation.

Within other aspects of the invention, the starting T cells are selectedfor negative or low expression of the T cell differentiation markerCD49d. Typically, CD49d expression is low or undetectable in naïve Tcells, and comparably high in Tem cells. Within these embodiments, aCD49d− phenotype will be marked by CD49d expression at levels equal to,or slightly (e.g., 10-20%) to moderately (20-45%) increased from, levelsof CD49d expression characteristic of naïve T cells and/or Tcm cells,and substantially lower than (e.g., less than 50% compared to) CD49dexpression levels found in Tem cells. These CD49d− T cells for usewithin the invention may be harvested from primary lymphoid organs ofanimals immunized with cognate Ag. CD49d− T cells most commonly used asstarting T cells express a CD49d− phenotype as described above, but mayotherwise depart phenotypically from activated naïve T cells and/or Tcmcells in expression of one or more other markers indicative of naïve Tcell and/or Tcm cell development, activation, and/or differentiation.

Within other aspects of the invention, the starting T cells are selectedfor negative or low expression of the T cell differentiation markerCD40L. Typically, CD40L expression is low or undetectable inunactivated, naïve T cells, and is rapidly upregulated upon T cellactivation to become comparably high in Tem cells. Within theseembodiments, a CD40L− phenotype will be marked by CD40L expression atlevels equal to, or slightly (e.g., 10-20%) to moderately (20-45%)increased from, levels of CD40L expression characteristic of naïve Tcells and/or Tcm cells, and substantially lower than (e.g., less than50% compared to) CD40L expression levels found in Tem cells. TheseCD40L− T cells for use within the invention may be harvested fromprimary lymphoid organs of animals immunized with cognate Ag. CD40L− Tcells most commonly used as starting T cells express a CD40L− phenotypeas described above, but may otherwise depart phenotypically from naïve Tcells and/or Tcm cells in expression of one or more other markersindicative of naïve T cell and/or Tem cell development, activation,and/or differentiation.

Within other aspects of the invention, the starting T cells are selectedfor negative or low expression of the T cell differentiation markerLIGHT, a recently identified member of the TNF superfamily (see, e.g.,Morel et al., Eur. J. Immunol. 33:3212 -19, 2003). Typically, LIGHTexpression is low or undetectable in unactivated, naïve T cells, and israpidly upregulated upon T cell activation to become comparably high inTem cells. Within these embodiments, a LIGHT- phenotype will be markedby LIGHT expression at levels equal to, or slightly (e.g., 10-20%) tomoderately (20-45%) increased from, levels of LIGHT expressioncharacteristic of naïve T cells and/or Tcm cells, and substantiallylower than (e.g., less than 50% compared to) LIGHT expression levelsfound in Tem cells. These LIGHT− T cells for use within the inventionmay be harvested from primary lymphoid organs of animals immunized withcognate Ag. LIGHT− T cells most commonly used as starting T cellsexpress a LIGHT− phenotype as described above, but may otherwise departphenotypically from naïve T cells and/or Tem cells in expression of oneor more other markers indicative of naïve T cell and/or Tem celldevelopment, activation, and/or differentiation.

Within other aspects of the invention, the starting T cells arephenotypically characterized by exhibiting a negative or lowproliferative response to IL-7 and or IL-15 cytokines. Typically, naïveT cells and Tem cells exhibit a negative or low proliferative responseto IL-7 and or IL-15, whereas Tem cells exhibit a highly efficientproliferative response to IL-7 and IL-15 cytokines (see, e.g., Geginatet al., Pathologie Biologie 51:64-66, 2003. Within these embodiments,the starting T cells will exhibit a proliferative response to IL-7 andIL-15 that is equal to, or slightly (e.g., 10-20%) to moderately(20-45%) increased from, levels of proliferation exhibited by naïve Tcells and/or Tem cells in response to exposure to IL-7 and IL-15, andsubstantially lower than (e.g., less than 50% compared to) IL-7- and/orIL-15-induced proliferation levels exhibited by Tem cells. These IL-7-and/or IL-15-unresponsive T cells for use within the invention may beharvested from primary lymphoid organs of animals immunized with cognateAg. IL-7- and/or IL-15-unresponsive T cells most commonly used asstarting T cells exhibit a IL-7- and/or IL-15-unresponsive phenotype asdescribed above, but may otherwise depart phenotypically from naïve Tcells and/or Tem cells in expression of one or more other markers orproliferative response indices indicative of naïve T cell and/or Tcmcell development, activation, and/or differentiation.

Within additional aspects of the invention, the starting T cell used forproduction of the T cell hybrid is an Ag-specific T cell which has beenAg-stimulated in vitro by contacting the T cell with the cognate Ag. Thestarting T cell may be initially stimulated with Ag in vitro, orrestimulated after collection of the T cell from a mammalian hostimmunized with the cognate Ag. Restimulation in this context is notrequired, but one or more rounds of Ag stimulation after collection of Tcells from an immunized subject may be undertaken prior to fusion of theT cell with the mammalian fusion partner cell. Antigen stimulation invitro may be achieved with or without concurrent contact of the startingT cell with an APC.

The T lymphocytes which can be used for fusion with the mammalian cellfusion partner are not otherwise particularly limited. Examples ofuseful T cells include those obtained from the bone marrow, lymph nodes,spleen, thoracic ducts, tonsils, peripheral blood, and thymus. Mostoften the T cells will be obtained from the bone marrow, lymph nodes, orspleen, although for convenience and reduction of morbidity in humandonors, peripheral blood mononuclear leukocytes (PBLs) may be used. If Tcells that produce specific immunoregulatory agents or mediators ofcellular immunity are desired, normal T lymphocytes may be cultured in amedium containing an inducer that induces production of such agents ormediators prior to or after fusion. The concentration of inducer in themedium will depend upon the particular inducer and the cellconcentration.

T lymphocytes can be isolated and or purified by various separatingmethods which are known, such as conventional physical methods, chemicalmethods and the adherence method to surface membranes, and can be usedfor fusion in accordance with the method described herein.

Ag-specific T lymphocytes are most often obtained by immunizing amammalian host with the cognate Ag. The cognate Ag may be anaturally-occurring or synthetic peptide Ag (e.g., comprising a T cellepitope), a portion of a protein containing a peptide Ag, an entireprotein containing one or more Ags, or a cell, cellular component,(e.g., membrane preparation), substrate, vehicle, or carrier comprisingthe Ag. The Ag may comprise one or more T cell epitope(s), and mayinclude epitopes from different sources (e.g., a fusion protein orconjugate of multiple epitopes from the same, or different, protein,cell, or organism).

For immunization, the cognate Ag is administered to the mammaliansubject to elicit an Ag-specific immune response in the subject.Immunization can be achieved, for example, by intravenous orsubcutaneous injection of the Ag, optionally conjugated with ahaptenating compound or hapten, or coupled to a carrier, membrane, orcell surface, or by live or attenuated infection or inoculation of cells(e.g., viral or bacterial pathogens, cancer cells, etc.) comprising thesubject Ag in an immunogenic state. After a period followingimmunization sufficient for T cell activation, the lymphocytes may beisolated from the immunized host and enriched for antigen-specific Tlymphocytes prior to fusion, according to various well known methods.

Within alternate embodiments of the invention, a naïve population of Tcells can be removed and primed for Ag-specific response in vitro.Several strategies known in the art have been employed to enrichantigen-specific T lymphocytes. One strategy is to supply the requisitesignals for activation and/or clonal expansion in vitro. Under theseconditions, Ag-specific cells increase in number and irrelevant cellsare diluted or die out. After a single restimulation with antigen,frequencies of greater than 1:100 can be achieved. With preferentialfusion of activated T cells, specific hybrids can be as frequent as onein five. Starting T lymphocytes may optionally be exposed to antigen/APCstimulation, and/or helper factors prior to fusion. The latter will begenerated in cultures from T inducer cell stimulation. If these cellshave been depleted, they can be reconstituted or a source of helperfactor added. Supernatant from mitogen-stimulated T cells (from whichmitogen has been removed or inactivated) or from secondary mixedlymphocyte cultures are convenient sources of helper factors.

The T cells to be used in the fusion can also be alloreactive Tlymphocytes, activated, for example, in vitro by mixed lymphocytereaction (MLR). MLR involves a reaction in which lymphocytes from afirst (responder) strain are mixed with lymphocytes from a second(stimulator) strain. The stimulator strain bears allogeneic MHCmolecules, and is typically irradiated or treated with mitomycin C torender them incapable of dividing. After a suitable period ofincubation, the responder cells are tested for Ag-specific responseactivity against cells of the stimulator strain bearing allogeneic MHCmolecules.

Useful antigens for generation of Ag-specific starting T cells include,but are not limited to, peptides comprising an immunodominant epitopeassociated with a mammalian immune disorder. Among the contemplatedimmune disorders in this context are autoimmune diseases, inflammatorydisorders, allergic conditions, cutaneous immune disorders, transplantrejection conditions, and graft versus host disease (GVHD). Othercognate Ags of interest include tumor antigens, and antigens ofpathogenic agents (e.g., viral and bacterial pathogens).

Within more detailed embodiments of the invention, Ag-specific Tlymphocytes are generated against cognate Ags comprising immunodominantepitopes associated with an autoimmune disease. For example, Ag-specificT lymphocytes may be generated against a variety of reported, MHC classII-restricted, immunodominant Ags associated with multiple sclerosis(MS), rheumatoid arthritis (RA), insulin-dependent diabetes mellitus(IDDM), chronic beryllium disease, autoimmune uveitis, sarcoidosis,systemic lupus erythromatosis, myasthenia gravis, Pemphigus vulgaris,Sjogren's syndrome, Addison's disease, autoimmune hepatitis, Gravesdisease, inflammatory bowel disease/Crohn's disease, and celiac disease.Alternatively, Ag-specific T lymphocytes may be generated against avariety of reported, MHC class I-restricted, immunodominant Agsassociated with psoriasis, ankylosing spondylitis, Reiter's disease, anduveitis.

Exemplary aspects of the invention are directed toward the autoimmunedisease MS. Within exemplary embodiments, the cognate Ag comprises animmunodominant, MS-associated epitope selected from a protein, peptideor epitope(s) of MOG (see, e.g., Forsthuber et al., J. Immunol.167:7119, 2001; Vandenbark et al., J. Immunol. 171:127-33, 2003), MBP(see, e.g., Madsen et al., Nat. Genet. 23:343, 1999), and or PLP (see,e.g., Kawamura et al., J. Clin. Invest. 105:977, 2000). Otherprospective Ags that may be important in MS and therefore useful withinthe invention are described for myelin-associated glycoprotein (MAG),non-myelin nervous system antigens, including the S10013 protein, andglial fibrillary acidic protein (GFAP), and non-neural specificantigens, such as heat shock proteins (hsps, including the small hsp αBcrystalline), transaldolase, and 2′,3′-cyclic nucleotide3′-phosphodiesterase (CNPase) (see, e.g., Kerlero J. Autoimmunity11:287-99, 1998).

Exemplary cognate Ags comprising immunodominant T cell epitopesassociated with MS for use within the invention include, but are notlimited to huMOG-1-22, huMOG-35-55, huMOG-huMOG-1-22, huMOG-34-54,huMOG-63-87, huMOG-64-96, huMOG-92-106, murine (mu)-MOG-1-30,muMOG-35-55, muMOG-81-110, muMOG-91-110, rat (rt)-MOG-1-20, rtMOG-35-55,rtMOG-74-90, hu-MBP-85-99, hu-MBP-86-99, hu-MBP-87-99, guinea pig(Gp)-MBP-72-89, rt-MBP-72-89, and PLP-139-151.

Other known, cognate Ags for use in generating Ag-specific T cells, Tcell hybrids, and RTLs within the invention include, for example,proteins, peptides and specific immunodominant epitopes mapped andreported for type II collagen (Rosloneic et al., J. Immunol.160:2573-78, 1998; Andersson et al., Proc. Natl. Acad. Sci. USA95:7574-79, 1998; and Fugger et al., Eur. J. Immunol. 26:928-33, 1996),human cartilage Ag gp39 (Cope et al., Arthritis Rheum. 42:1497, 1999),and E. coli heat shock peptide that are associated with RA, and glutamicacid decarboxylase 65 (Patel et al., Proc. Natl. Acad. Sci. USA94:8082-87, 1997; Wicker et al., J. Clin. Invest. 98:2597, 1996),insulin (Congia et al., Proc. Natl. Acad. Sci. USA 95:3833-38, 1998),and insulinoma antigen that are associated with IDDM. Yet additionalcognate Ags for use in generating Ag-specific T cells, T cell hybrids,and RTLs within the invention include, for example, proteins, peptidesand specific immunodominant epitopes mapped and reported for Addison'sdisease (adrenocortical 21-hydroxylase), autoimmune hepatitis (anti-smcantibodies (Abs), anti-mitochondrial Abs, and anti-liver/anti-kidneyAbs), celiac disease (gluten peptides), Graves disease (TSH receptor),myasthenia gravis (acetylcholine receptor), pemphigus vulgaris(epidermal cadherin, keratinocyte cell surface antigens), and systemiclupus erythematosus (nucleoproteins).

In addition to the phenomenon of immunodominant peptide recognition, TCRutilization in autoimmune responses is often quite limited, despite avast diversity of TCRs available in the T cell population. For example,limited TCR utilization has been reported in the immune response ofLewis rats immunized with myelin basic protein (MBP) to induce EAE. TheT cell response thus induced is directed primarily to an immunodominantepitope contained within an encephalitogenic fragment of MBP comprisingamino acids 66-88 of the protein (MBP 66-88). The T cell response isalso highly restricted, dominated by T cells expressing TCR Vβ8 almostexclusively and Vα2 frequently (Burns et al., J. Exp. Med. 169:27,1989). Similarly, Gold et al., J. Exp. Med. 174:1467-76, 1991 reportconserved TCR Vα and Vβ utilization in EAE, wherein the TCR β chainsequences of T cell clones and hybrids reactive to MBP 68-88 allutilized a Vβ8.2 segment, and exhibited other conserved structuralfeatures.

In accordance with the limited TCR repertoire involved in autoimmunity,and in relation to other desired attributes and features of theinvention, the T cell hybrids described herein will typically possessdefined TCR structural and functional features. In certain embodiments,the starting T cell will express one or more TCR gene(s) encoded by atransgene (i.e., including genes introduced into animal germline cellsby transgenesis, and recombinantly engineered and expressed genesintroduced into cells by recombinant expression vectors yieldingtransformation of the recipient cells to express the transgenic orrecombinant TCR). In related aspects, one or more recombinant TCRgene(s) may be introduced into the starting T cell or hybrid bytransduction or transfection using a recombinant expression vector thatencodes the TCR gene(s) of interest. Typically, the subject TCR genewill be a TCR that specifically binds a cognate Ag (for example an Agassociated with a T cell autoimmune response or activity) and/ormediates T cell activation in an Ag-specific manner.

For methods and composition directed toward TCR structure-functionanalysis and rational design (i.e., including TCR design, engineering,construction, screening and characterization) the T cell or T cellhybrid may be express all or part of a TCR α chain, a TCRβ chain, a TCRγchain, and/or a TCRΔ chain. Often, the TCR transgene or recombinantvector will be introduced into a RAG-deficient or RAG-null Tg mammaliansubject or transformed recipient cell. For example, the TCR-encodingvector may be transduced or transfected into a mammalian cell that has amutation in a RAG-1 and/or RAG-2 gene that renders the cell null ordefective for RAG-1 and/or RAG-2 expression (see, e.g., Katz, et al.,Cell 74:1089 1100, 1993; Gonzalez et al., Immunity 7:873 883, 1997).

In more detailed embodiments, the one or more TCR gene(s) that may beintroduced transgenically or recombinantly into a subject or target cellfor TCR expression will encode a selected TCR segment, chain, orcomplete receptor. TCR components of interest for expression in thisregard include TCR components identified, for example, as mediatingAg-specific responses of T cells directed toward an Ag of interest, forexample an immunodominant epitope associated with an autoimmune disease.Exemplary TCRs include TCRs, TCR segments, and TCR domains encoded by aselected AV or BV gene. For example, transgenic or recombinant TCRs ofinterest may include a TCR, segment, or domain encoded by a BV8, Vα2AV11, or AV17 gene. Any AV or BV gene/gene product can be adapted inthis context for use within the method and compositions of theinvention.

In various exemplary embodiments, TCRs may be cloned from autoreactive Tcells or T hybrids and further engineered for use within the foregoingmethods and compositions. Often, the TCR will be engineered toincorporate modifications for structure-function analyses, for examplethe TCR may be modified at a putative Ag- or MHC-interactive domain orbinding interface to incorporate site-directed mutations to develop andmap novel receptor target sequences (e.g., for development of TCRagonists and antagonists). Various methods for TCR identification,cloning, mutation, recombinant expression, structure-function mapping,and screening (e.g., to screen small molecule drugs that modulate TCRbinding or activation) are well known and widely practiced in the art(see, e.g., Pankewycz et al., Eur. J. Immunol. 21:873 879, 1991; Katz etal., Cell 74:1089-1100, 1993; Daniel et al., Eur. J. Immunol. 25:10561062, 1995; Haskins et al., Diabetes 37:1444 1448, 1988; Haskins et al.,Proc. Natl. Acad. Sci. USA 86:8000 8004, 1989; Haskins et al., Science249:1433 1436, 1990; and Nakano et al., J. Exp. Med. 173:1091-1097,1991; and Gonzalez et al., Immunity 7, 873 883, 1997).

Exemplifying these complementary methods and tools for use within theinvention, Strattman et al., (J. Clin. Invest. 112:902-14, 2003)describe isolation and transgenic expression of TCRs from anautoreactive, BDC-2.5 T cell line derived from a diabetic female NODmouse. This cell line is a CD4+ Th1 clone that displays a Vβ4/Vα1(AV1S5) TCR heterodimer (Candeias et al., Proc. Natl. Acad. Sci. USA88:6167-6170, 1991) and was shown to greatly accelerate disease whentransferred to young animals. Mice transgenic for the BDC-2.5 TCR wereproduced and employed by Strattman et al. for various purposes in theirstudy. The cDNA for the α and β chains of the BDC-2.5 TCR was obtainedby RT-PCR according to known methods. PCR products were subcloned into asuitable cloning vector, sequenced, and subcloned into a metallothioneinpromoter-based fly expression vector. Each of the final constructs codedfor the α1α2 and the β1β2 domains, respectively, followed by a linkersequence (SSADL), a thrombin site

(LVPRGS), a leucine zipper (acidic for the a chain, basic for the βchain), and a hexahistidine tag. Vectors were transfected into SC2cells, and stable cell lines and clones were established. Soluble TCRswere purified from culture supernatants according to known methods. Inaddition, Strattman et al. describe useful methods for cell preparation,cell staining, flow cytometry analysis, and adoptive transfer. In onemodified assay, Strattman et al. constructed an MHC-mimetic peptide withhigh agonistic activity for BDC-2.5 T cells, which was complexed to theAg7 molecule in an MHC multimer molecule (Crawford et al., Immunity8:675 682, 1998; Altman et al., Science 274:94 96, 1996) and assayedagainst. Tetramers of the Ag7/mimotope complex were shown to be specificfor BDC-2.5 T cells.

In the same context, the invention provides for expression of transgenicand recombinant TCRs by animals (e.g., animals from which starting Tcells are obtained for fusion), by T cells, and by T cell hybrids thatmay be assayed (e.g., for TCR binding and/or activation) against TCRligands, including native and/or recombinant ligands (e.g., RTLs, seebelow), and these materials and methods can be used in screens toidentify other compounds (e.g., peptides and small molecule drugs) thataffect Ag-specific TCR binding and/or activation) Further detailsregarding the methods and tools that can be employed in this aspect ofthe invention are provided elsewhere herein and/or among the referencescited herein, which are all incorporated herein by reference). Accordingto one exemplary procedure, Strattman and colleagues produced afunctional transgenic BDC-2.5 TCR mice and transformed transgenicBDC-2.5 TCR T cell hybrids, as well as an Ag7/2.5 mimotope complex. Thissynthetic ligand for the BDC-2.5 T cells was isolated from a chemicallysynthesized random-peptide library based on its ability to stimulate theBDC-2.5 T cells (as measured by IL2 assay). Alternatively, the TCRtransfectants were screened for surface expression of TCR and verifiedto be functional as determined by release of IL-2 after stimulation withan appropriate VP-selective superantigen (presented by autologousEpstein-Barr virus-transformed B cells). In a related report, Fan etal., Proc Natl Acad Sci USA 100: 3386-91, 2003, constructed TCR Tg micethat provide a model of inflammatory skin disease, in whichkeratinocytes activate and are the primary target of autoreactive CD4+ Tcells. In particular, the investigators generated keratin 14 (K14)-Aβbmice expressing MHC class II only on thymic cortical epithelium, andshowed that the CD4+ T cells from these K14-Aβb mice fail to undergonegative selection and thus have significant autoreactivity. The TCRgenes from an autoreactive K14-Aβb CD4 T cell hybridoma were cloned toproduce the TCR Tg mouse, and the Tg cells were negatively selected inWT C57BL/6 mice but not in 2-2-3/K14-Aβb mice. Additional methods andtools for use within these aspects of the present invention aredescribed, for example by Laufer et al. (Nature 383:81-85, 1996), Martinet al. (Cell 84:543-50, 1996), Laufer et al. (J. Immunol. 162:5078-84,1999), (Berg et al., Mol. Cell. Biol. 8:5459-69, 1988), Ho et al., (J.Exp. Med. 179:1539-49, 1994), Rojo et al., (J. Immunol. 140:1081-88,1988), Kersh et al. (J. Immunol. 161:585-93, 1998), Haskins et al.(Diabetes 37:1444-48, 1988), Bensinger et al. (J. Exp. Med. 194:427-38,2001), Wells et al. (J. Clin. Invest. 100:3173-83, 1997), and Vassar etal. (Genes Dev. 5:714-27, 1991).

[1 06] In accordance with other aspects of the invention, the T cellhybrids described herein will typically possess defined MHC structuraland functional features. In certain embodiments, the starting T cellwill express one or more native or recombinant (including wild type orselected mutant) MHC gene(s) encoded by a transgene. In related aspects,one or more recombinant MHC gene(s) may be introduced into the startingT cell or hybrid by transduction or transfection using a recombinantexpression vector that encodes the MHC gene(s) of interest. Typically,the subject MHC gene will be a MHC II gene that encodes a portion of anMHC II complex that specifically binds a cognate Ag (for example an Agassociated with a T cell autoimmune response or activity) and/ormediates T cell activation in an Ag-specific manner.

For methods and composition directed toward MHC structure-functionanalysis and rational design the T cell or T cell hybrid may express allor part of a MHC I or MHC II molecule, for example a MHC II α chain, orMHC II β chain. Alternatively, the T cell or T cell hybrid may express arecombinant MHC molecule, including recombinant MHC molecules comprisingone or more selected portions or domains of an MHC chain. In exemplaryembodiments, the subject recombinant MHC molecule may comprise a singlechain MHC II construct including selected, peptide binding and/orTCR-interactive portions of the corresponding native MHC II molecule(e.g., as described herein for single chain MHC II molecules associatedor bound with cognate Ag peptides, comprising an RTL).

Within more detailed embodiments of the invention, the starting T cellis transgenic for a selected MHC genotype. Typically, these starting Tcells are obtained from a MHC Tg subject. Exemplary MHC Tg subjectsinclude a large assemblage of Tg murine subjects known and available inthe art that are Tg for a selected MHC class II (MHC II) genotype.Commonly, the selected MHC II genotype will be a mutant MHC II isotypeassociated with an immune disorder, frequently an autoimmune disease.The MHC II genotype will typically specify an MHC II that functions torestrict Ag-specific, TCR-mediated immune responses. Exemplary T cellhybrids of the invention will therefore often be constructed using astarting T cell that is transgenic for a mutant HLA-DR, HLA-DP, orHLA-DQ isotype that contributes to a mammalian immune disorder, forexample an autoimmune disease, inflammatory disorder, allergiccondition, cutaneous immune disorder (psoriasis, atopic dermatitis,cutaneous T cell lymphoma), transplant rejection condition, or graftversus host disease (GVHD). Numerous such isotypes are known andcharacterized in the art in association with autoimmune disorders,including MS, RA, IDDM, chronic beryllium disease, autoimmune uveitis,sarcoidosis, systemic lupus erythromatosis, myasthenia gravis, andceliac disease.

In related embodiments, the mutant HLA-DR, HLA-DP, or HLA-DQ isotype isselected from a mutant HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DP2,HLA-DQ6, or HLA-DQ8 isotype. Exemplary mutant MHC II isotypes in thiscontext include, but are not limited to, DRB1*0101; DRB1*0301,DRB1*0401, DRB1*0405, DRB1*1501, DRB4*0101, DRB5*0101, DPA1*0101,DPA1*0103, DPB1*0201, DPB1*0401, DQA1*0102, DQA1*0501, DQB1*0201,DQB1*0301, DQB1*0302, DQB1*0602, and DQB1*0604.

Among the features of these novel T cell hybrid expressing a MHC IItransgene (and/or a selected mutant MHC isotype), the Ag-specific,TCR-mediated proliferative response of these hybrid will typically berestricted by the corresponding HLA-DR, HLA-DP, or HLA-DQ MHC II.

For constructing T cell hybrids of the invention, a variety of usefulfusion partner cells are provided. Typically, the mammalian fusionpartner cell is from a tumor or other cancer cell line. Most commonlythe fusion partner cell is a clonal myeloid or T cell line. In alternateembodiments, the mammalian fusion partner cell or the T cell hybrid istransformed into a clonal cell line by transduction or transfection ofan immortalizing gene into said cell. Commonly used immortalizing genesin this context include various oncogenes, such as ras. In relatedembodiments, the mammalian fusion partner cell or the T cell hybrid istransformed into a clonal cell line by viral transformation, for exampleby Epstein Barr virus (EBV) transformation (see, e.g., U.S. Pat. No.4,720,459, issued Jan. 19, 1988).

The starting T cells and the mammalian fusion partner cells forgenerating the T cell hybrids may be from any mammalian species and maybe from the same species or two different species in a given fusion.Commonly, both the starting T cell and mammalian fusion partner cellwill be of the same species, for example both human or both murinecells. Typically the fusion partner cell will be deficient or null forTCR expression, for example they will not exhibit surface expression ofintact, functional TCRs, or they will be genotypically null for one ormore TCR genes.

In more detailed embodiments, the starting T cells are obtained frommurine, rat, rabbit, or other suitable non-human, mammalian subject.Commonly, the fusion partner will correspond to the starting T cell inspecies and/or individual origin. In exemplary embodiments, the T cellhybrid is formed by fusion of a murine T cell, typically from a Tgmurine background, with a murine T cell line. Various murine T cellcancer lines (e.g., lymphoma and thymoma lines) are useful within thiscontext. In exemplary embodiments, a variant of the well known murinethymoma line BW5147 is employed as the fusion partner. For T cellfusions, the BW5147 thymoma is the most widely used tumor partner cellline (Chien et al., Nature 312:31-35, 1984; Yague et al., Cell 42:81-87,1985; Blackman et al., Immunol. Rev. 101:5-19, 1986; Lee et al., J.Immunol. 140:1665-75, 1988). Although this tumor cell line does not bearsurface TCR, existing endogenous TCR genes can be expressed as surfacereceptors after fusion with a normal T cell (Yague et al., Cell42:81-87, 1985; Blackman et al., Cell 47:349-57, 1986). White et al.isolated two variants of the BW5147 cells that can no longer expressfunctional TCR α.- and β-chains (White et al., J. Immunol. 143:1822-25,1989). These variants have been used to determine the structure of TCR δchain (Born et al., Nature 330:572-74, 1987), and to provide anappropriate tool for analysis of the TCR repertoire used in response toany given Ag, including self-determinants (Blackman et al., Immunol.Rev. 101:5-19, 1988; Kappler et al., Nature 332:35-40, 1988). SuitableBW5147 variants for use within the invention will often be aTCR-deficient or null BW5147 variant. For example, a desired BW5147variant may be negative for cell surface expression of TCRα and TCRβ.

To further facilitate production and use of the T hybrids of theinvention, mammalian fusion partner cells will most commonly be selectedthat are deficient or null for CD3 and/or CD4 expression. In certainembodiments, the fusion partner will not detectably express one or bothof the CD3 and/or CD4 products.

In other detailed embodiments, the starting T cells are human T cells,and the mammalian fusion partner cell is a human T leukemia line. The Tleukemia cell line is commonly a AH-sensitive mutant derived from theJurkat human T leukemia cell line. The derivation of the Jurkat line wasoriginally achieved by successively culturing parent Jurkat cells inmedia containing graded concentrations of 6-thioguanine. Prior toculturing, the parent cells may be optionally exposed to a mutagenicagent, e.g., an alkylating agent such as ethylmethane-sulfonate, toincrease the frequency of mutants. Surviving cells exhibit an inabilityto incorporate 6-thioguanine suggesting they are deficient inhypoxanthine-guanine phosphoribosyl transferase (HGPRT).

An exemplary Jurkat T leukemia line for use within the invention is theJ3R7 T leukemia cell line deposited at the American Type CultureCollection, (ATCC), Manasass, Va. on 2 Sep., 1982, which has beenassigned the ATCC number CRL 8169. Other examples of well known,publicly available human cell lines for use as immortalizing fusionpartners with T cells include human KE37.3.2, KE 37, MOLT3, MOLT 4,CCRF-CEM, and HuT 78 cells (see, e.g., U.S. Pat. No. 4,665,032, issuedMay 12, 1987).

To construct a parent leukemia cell line, the cells may be cultivated insuccessive incubations using, for example, graded concentrations of6-thioguanine. The initial 6-thioguanine concentration is preferablyabout 10⁻⁵ M and is progressively increased to about 10⁻⁴ M. The totalincubation time will typically be about 6 to about 12 weeks with themedium changed weekly. Accordingly, the number of incubations willusually be about 6 to 12. All incubations are carried out atapproximately physiological temperature (e.g., about 37° C.).Conventional nutrient media such as RPMI 1640 may be used in thesecultivations supplemented with 10% fetal calf serum (FCS). Eachincubation will usually take about one week, with viable cells beingisolated before the succeeding incubation. Sufficient cells are used ineach incubation to ensure the likelihood of obtaining viable cells atthe end of the incubation period. The number of cells per flask (25 ccvolume) will usually be about 10⁷. The fastest growing 6-thioguanineresistant clones are chosen for use in the fusions. Such clones willtypically have a doubling time in the range of about 18 to 24 hr. Ifnecessary, the clones may be expanded prior to fusion to provide asuitable supply of cells for the fusion.

Fusion of the starting T cells and mammalian cell fusion partners can becarried out according to various well-known methods. Typically, the Tcell and the mammalian fusion partner cell are fused to form the T cellhybrid by contacting the T cell and the fusion partner cell in thepresence of a fusogen. In exemplary embodiments, the fusogen is apolyethylene glycol (PEG), for example PEG 1500 (Roch Diagnostics,Mannheim, Germany). Alternatively, the fusogen may be a virus, which canbe any of a number of well known fusogenic viral strains, for exampleSendai virus.

To accomplish the T cell fusions, a ratio of starting T cells to fusionpartner cells can vary according to standard protocols. After fusion,unfused fusion partner cells die off after a few days in culture, andthe fused cells can be separated from the unfused starting T cells byvarious known methods. In one exemplary method, the post-fusion cellmixture containing the fused as well as the parental cells may beincubated in a medium containing the selective reagent for a period oftime sufficient to eliminate most of the unfused cells. For instance, anumber of tumor cell lines are sensitive to HAT due to lack offunctional hypoxanthine-guanine phosphoribosyl transferase (“HGPRT”).Fused cells formed by starting T cells and these tumor cell lines becomeresistant to HAT, as the starting T cells contribute functional HGPRT.Thus, a HAT selection can be performed after fusion to eliminate unfusedparental cells.

In the event that the fused T hybrid cells lose certain starting T cellcharacteristics, such as expression of a selected TCR, they (i.e.,primary fused cells) can be refused with the same or different startingT cells to restore a desired phenotype. Such refused cells (i.e.,secondary fused cells) may be particularly potent or sensitive withinthe methods and compositions of the invention.

Although HAT fusion is a useful fusion and selection within theinvention, alternative processes may be employed that avoid potentialgrowth inhibitory effects of thymidine in HAT medium. Fox, et al.,(Cancer Res 40:1718-21, 1980) report that the inhibitory effect of thethymidine can possibly be alleviated by the addition of deoxycytidine.Alternatives to HAT, such as a hypoxanthine-azaserine-thymidine mediumhave been used in B cell fusions to increase the yield of hybrids. Morerecently, Engleman et al. (U.S. Pat. No. 4,950,598, issued Aug. 21,1990) reported fusing a HAT-sensitive human T leukemia line with anormal human T lymphocyte to produce human T cell hybridomas that werestable and produced IL-2 constitutively. In addition Engleman et al.,reported that T cell fusions could be improved by fusing anazaserine-hypoxanthine sensitive human T leukemia (Jurkat) cell linewith normal human T cells and culturing the hybrids in a selectiveazaserine-hypoxanthine medium—avoiding potential toxic effects on growthattributed to thymidine-containing selective media. Cultures ofsubclones from these fusions also reportedly exhibited stable,continuous growth and continuous production of IL-2.

For use within the invention, AH-sensitive T leukemia cells and starting(normal) T lymphocytes are fused by combining the two in a mediumcontaining a fusogen. Polyethylene glycol (PEG) of about 1500 daltons isa preferred fusogen, but other PEGs of molecular weight in the range ofabout 1000 to 4000 daltons or other fusogens may be used. The fusogenwill normally be present at between about 40% and 60% (v/v), preferablyat about 50% (v/v), in the medium. The medium is preferably free ofserum. The starting T cells and T leukemia cells will usually becombined at ratios of 2:1 to 1:10 with the total cells each being in therange of about 10⁶ to 5×10⁷. A volume of 0.5 to 1 ml of fusion mediumper 10⁸ cells is often used. The cells are normally exposed to thefusion medium for about 5 to 10 minutes, often for about 7 to 8 minutes.A preferred fusion technique involves suspending the cells in the fusionmedium followed by centrifugation of the cells while in contact withfusion medium. The fusion may be carried out at room temperature,typically with the fusogen prewarmed to 37° C.

Following the fusion the cells are separated from the fusion medium andwashed repeatedly with fusogen-free medium to remove residual fusogen.The cells are then resuspended in the selective AH medium. The AH mediumconsists of a standard cell culture medium, such as RPMI or Dulbecco'smedium, containing about 1 to about 20, preferably about 10, .μgazaserine per ml. The concentration of hypoxanthine in the medium isabout 100 μM. The medium will also preferably contain serum, typicallyFCS, at a level of about 10% to 15% (v/v), an appropriate antibioticsuch as penicillin/streptomycin and L-glutamine. Azaserine is a diazoanalog of L-glutamine. Its main effect is an irreversible binding tovarious L-glutamine amino-transferases which are necessary in de novopurine biosynthesis. Its effect on pyrimidine synthesis is minimal andreversible with the addition of ademine. The use of azaserine in theselective medium avoids the necessity of using aminopterin which is ananti-folic acid antagonist and inhibits both purine and pyrimidinesynthesis. Therefore, the use of azaserine eliminates the need forthymidine as an exogenous source of pyrimidine in the medium. Thisresults in increased fusion efficiency and an increased rate of hybridgrowth.

The success of T cell fusions and the stability of the resulting hybridsmay be verified by a variety of known methods, for example includingchromosomal analysis of the hybrids by conventional fixation andstaining techniques. The resulting stable hybrid cells may be grown invitro or in vivo by various known techniques. Production ofimmunoregulatory agents or mediators may be enhanced in the hybrids byadding inducers to the culture medium. In vivo cell growth may beeffected by introducing the hybrid clones into a suitableimmunodeficient host, such as nude mice or heavily irradiated mice. Theclones will grow as ascites-producing tumors in such hosts. Compoundsproduced by the hybrids may be isolated from the culture medium, ascitesfluid, or serum, as the case may be, by known separation andpurification techniques such as ammonium sulfate precipitation,dialysis, chromatography, and gel electrophoresis.

The T cell hybrids of the present invention are useful in the analysisof the properties of individual T cells, as well as the underlyingcellular and molecular events important in their activation. Forexample, the hybrids are well adapted for analyzing individual T cellspecificities. In addition, the hybrids can be used to identify andcharacterize molecular events and constituents involved in T cellactivation, including TCR binding and activation, and TCR-mediated Tcell activation responses. For example, class II restricted T cellhybrids of the invention are useful for identifying, mapping, andcharacterizing determinants on cognate Ags, and on synthetic analogs andderivatives of cognate Ags, including peptide components of RTLs.Likewise, class II restricted T cell hybrids of the invention are usefulfor identifying, mapping, and characterizing determinants binding andactivation sites or domains on TCRs that are critical to Ag-specific Tcell activation. The hybrids are also useful for determining the rolesof MHC molecules, and for mapping the structural and functional domains(e.g., peptide and TCR interactive sites) on MHC molecules and MHCcomponents of RTLs, involved in antigen processing, antigenpresentation, and/or MHC-TCR interactions.

In addition, the present invention provides for a broad range ofmanipulations of the hybrid cells, for example culturing,Ag-stimulation, and exposure to test immune modulatory agents underdefined conditions, transformation of the hybrid cells (or starting Tcells or fusion partner cells), for example, to express immunemodulatory molecules, costimulation regulatory molecules,neuroprotective and/or neuroregenerative factors, growth factors,transgenic or recombinantly-modified TCRs, and exposure of the hybridcells to prospective immune modulatory agents, for example smallmolecule drugs and RTLs (e.g., having novel or modified peptide and/orMHC components) to screen and characterize TCR-modulatory and/or T cellmodulatory agents.

One very promising approach for regulating antigen-specific T cellresponses in autoimmunity and in other contexts (e.g., graft rejection)is to reprogram or induce nonresponsiveness in T cells using recombinantor synthetic TCR ligands, or T cell modulatory drugs or other compoundsthat are agonists or antagonists for activation of TCRs by their cognateligands. In this regard, various analogs of TCR ligands have beenproduced which comprise extracellular domains of class II MHC moleculeslinked to specific peptide targets. Several such constructs have beendeveloped that involve natural or recombinant α1α2 and β1β2 MHC class IIdomains in association with various encephalitogenic or other pathogenicpeptides covalently linked or noncovalently bound to the MHC IIcomponent to form a complex (Kozono et al., Nature 369:151, 1994;Fremont et al., Science 272:1001, 1996; Sharma et al., Proc. Natl. Acad.Sci. USA 88:11405, 1991; Nicolle et al., J. Clin. Invest. 93:1361, 1994;Spack et al., CNS Drug Rev. 4:225, 1998). These molecular complexes bindnot only to the TCR but also to the CD4 molecule on the T cell surfacethrough the β2 MHC domain (Brogdon et al., J. Immunol. 161:5472, 1998),and have been reported to inhibit T cell activation and prevent EAE inrodents (Sharma et al., Proc. Natl. Acad. Sci. USA 88:11405, 1991; Spacket al., CNS Drub Rev. 4:225, 1998; Steward et al., J. Allerg. Clin.Immun. 2:S117, 1997).

An even more promising design for TCR modulatory agents in this contextare recombinant T cell receptor ligands (RTLs) that incorporate aminimal TCR interface, for example comprising only the al and 131 MHCdomains (or otherwise excluding the β2 CD4-binding domain) covalentlylinked to peptide (Burrows et al., Prot Eng. 12:771, 1999). These RTLconstructs have been shown to prevent and treat MBP-induced EAE in Lewisrats (Burrows et al., J. Immunol. 161:5987, 1998; Burrows et al., J.Immunol. 164:6366, 2000) and to inhibit activation and induce IL-10secretion in human DR2-restricted T cell clones specific for MBP-85-95or BCR-ABL b3a2 peptide (CABL) (Burrows et al., J. Immunol. 167:4386,2001; Chang et al., J. Biol. Chem. 276:24170, 2001). Additional RTLconstructs have been designed and tested by inventors in the instantapplication, which include a MOG-35-55/DR2 construct (VG312) shown topotently inhibit autoimmune responses and lead to immunologicaltolerance to the encephalitogenic MOG-35-55 peptide and reverse clinicaland histological signs of EAE (Vandenbark et al., J. Immunol.171:127-33, 2003). Numerous additional RTL constructs that are usefulfor modulating T cell immune responses and can be employed within theinvention are available for use within the methods and compositions ofthe invention (see, e.g., U.S. Pat. No. 5,270,772, issued Aug. 7, 2001;U.S. Provisional Patent Application No. 60/064,552, filed Sep. 16, 1997;U.S. Provisional Patent Application No. 60/064,555, filed Sep. 16, 1997;U.S. Provisional Patent Application No. 60/200,942, filed May 1, 2000;United States Provisional Patent Application entitled MONOMERICRECOMBINANT MHC MOLECULES USEFUL FOR MANIPULATION OF ANTIGEN-SPECIFICT-CELLS, filed b_(y) Burrows et al. on Sep. 5, 2003 and identified byAttorney Docket No. 49321-98; U.S. patent application Ser. No.09/153,586; filed May 1, 2001; U.S. patent application Ser. No.09/847,172; filed May 1, 2001; and U.S. patent application Ser. No.09/858,580; filed May 15, 2001, each incorporated herein by reference).

To evaluate the biological function and mechanisms of action of RTLs andother T cell modulatory agents, antigen-specific T cells bearing cognateTCRs have been used as target T cells for testing (see, e.g., Burrows etal., J. Immunol. 167:4386, 2001). However, a low frequency ofAg-specific T cells, varying levels of T cell Ag-specific responses, anda potential for uncontrolled interactions (e.g., with other, differentcells) have significantly limited the scope of these investigations.

The T cell hybrids of the invention are uniquely adapted for use inscreens and assays to identify and characterize RTL structure-function.To practice these aspects of the invention, T cell hybrids areconstructed and selected that display an Ag-specific, TCR-mediatedproliferative response contact of the hybrid with a cognate Ag and APCs.This proliferative response of T hybrids can in turn be detectablyinhibited or stimulated by contacting the T cell hybrid with an RTL ofinterest, which yields a modified, Ag-specific, TCR-mediatedproliferation response of the hybrid. The modified proliferationresponse of the hybrid cell accurately and reproducibly indicates apresence, quantity, and/or activity level of the RTL in contact with theT cell hybrid.

RTLs for use in these aspects of the invention will often comprise a MHCII component covalently linked or non-covalently bound to the cognateAg. In exemplary embodiments, the RTL comprises a single chain MHC IIcomponent comprised of MHC II al and 131 domains. Alternatively, the RTLmay comprise other single chain MHC II components which lack a β2 orother CD4-interactive domain. RTLs of interest in these aspects of theinvention include RTLs which have the cognate Ag covalently linked ornon-covalently bound to the MHC II component, wherein the cognateantigen comprises an immunodominant T cell epitope associated with amammalian immune disorder, for example an autoimmune disease. Inexemplary embodiments, the autoimmune disease is MS, and theimmunodominant epitope is selected from human (hu)-myelinoligodendrocyte protein (MOG)-1-22, huMOG-35-55, huMOG-huMOG-1-22,huMOG-34-54, huMOG-63-87, huMOG-64-96, huMOG-92-106, murine(mu)-MOG-1-30, muMOG-35-55, muMOG-81-110, muMOG-91-110, rat(rt)-MOG-1-20, rtMOG-35-55, rtMOG-74-90, guinea pig (Gp)-myelin basicprotein (MBP)-72-89, rt-MBP-72-89, hu-MBP-85-99, hu-MBP-86-99,hu-MBP-87-99, PLP-139-151. In more detailed embodiments, the RTL isVG1000, which includes a covalently bound hMOG-35-55 Ag.

In related embodiments of the invention, the RTL is modified to reducehigher order aggregate formation by the RTL. Such modifications includemutations in a MHC component of the RTL, for example by recombinantintroduction of one or more mutations within a B sheet platform of anMHC II component.

In addition to Ag-specific TCRs and MHCs, a number of other regulatorymolecules regulate T cell activation and impart sensitivity andplasticity to the immune response. Within the methods and compositionsof the invention, it will be additionally useful to express one or moresuch regulatory molecules within the starting T cells, mammalian fusionpartner cells, and/or the T cell hybrids described herein. In certainembodiments of the invention, the starting T cell, mammalian fusionpartner cell, and/or the T cell hybrid is transgenically modified,transduced, or transfected to express an immune modulatory molecule.Often, the immune modulatory molecule will be a cytokine, for exampleIL-2. Another immune modulatory molecule of interest is the product ofFox P3, which may be expressed in the starting T cell or hybrid to drivea T suppressor phenotype or function in the hybrid cell.

In additional embodiments, the immune modulatory molecule is a T cellcostimulation regulatory molecule, or an agonist, antagonist, or ligandof a T cell costimulation regulatory molecule. Exemplary modulatorymolecules in this context include, OX 40, OX 40 ligand, CD 40, CD 40ligand, CD 28, CD 28 ligand, CTLA4, and CTLA4 ligand.

In yet additional embodiments of the invention, the starting T cell, themammalian fusion partner cell, and/or the T cell hybrid istransgenically modified, transduced, or transfected to express aneuroprotective or neuroregenerative factor. Exemplary neuroprotectiveor neuroregenerative factors in this context include nerve growth factor(NGF) and brain-derived neural factor (BDNF).

In still other embodiments, he T cell, the mammalian fusion partnercell, and/or the T cell hybrid is transgenically modified, transduced,or transfected to express a growth factor, for example IL-7 or IL-15.

The T cell hybrids of the invention are particularly well suited forscreening, designing, and characterizing T cell modulatory agents fordevelopment as diagnostic and/or therapeutic agents in the managementand treatment of multiple sclerosis (MS). MS is a chronic autoimmunedisease characterized by recurrent attacks of neurologic dysfunction dueto lesions in the central nervous system. MS lesions are marked byinfiltration of inflammatory cells, such as lymphocytes, macrophages andneutrophils, and associated axonal demyelination (Martin et al., Crit.Rev. Clin. Lab. Sci. 32:121-82, 1995; Steinman, L., Nat. Immunol.2:762-65, 2000). The classic clinical features of multiple sclerosisinclude impaired vision and weakness or paralysis of one or more limbs.After a number of years, patients generally experience a progressivedeterioration of neurologic function. The disease course isunpredictable and involves exacerbations and remissions in 75% ofpatients. Although a few patients die within the first few years ofonset, the average duration of the disease is greater than 30 years.

There are an estimated 250,000 cases of multiple sclerosis in the UnitedStates, with approximately 10,000 new cases occurring each year. Thecause of MS is unknown but epidemiology implicates immunologic orinfectious factors resulting in a chronic inflammatory condition.Multiple sclerosis is typically a disease of young adults, with morethan 65% of cases initiating at ages 20-40. 60% of MS patients arewomen. Over one million physician visits occur annually for multiplesclerosis in the United States alone.

There is currently no effective treatment or cure for multiplesclerosis. MS therapy is directed toward reducing the severity of acuteepisodes and preventing relapses. In acute flare-ups, steroids are oftenemployed to reduce severity and speed recovery. Experimental therapywith other immunosuppressive agents, such as cyclophosphamide, has alsobeen attempted, but with limited success.

Both genetic and environmental factors have been implicated in the MSdisease process. Genetic predisposition to MS includes an association ofthe disease with certain class II major histocompatibility complex (MHC)haplotypes, in particular HLA-DR2 (DRB1*1501) and DQw1 (Ho et al.,Immunogenetics 15:509-17, 1982; Hauser et al., Neurology 39:275-77,1989; Zipp et al., Hum. Immunol. 61:1021-30, 2000).

A widely accepted disease model for human MS is experimental allergicencephalomyelitis (EAE), an acute inflammatory and demyelinating diseaseof the central nervous system (CNS) bearing substantial clinical andpathological similarities to MS (Traugott et al., Science 219:308-10,1983; Zamvil et al., Nature (London) 317:355-58, 1985; Rose, et al.,Clin. Immunol. Immunopathol. 59:1-15, 1991. For both EAE and MS, thereis considerable evidence that immunological and inflammatory processescontribute to the pathogenesis of the disease. (Hauser, et al., AnnNeurol. 13:418-25, 1983; Traugott, et al., Cell. Immunol. 68:261-75,1982; Rose, et al., Clin. Immunol. Immunopathol. 45:405-23, 1987). Thisis supported by the presence of perivascular mononuclear cellularinfiltrates in MS lesions and macrophage-dependent phagocytosis ofmyelin in the CNS white matter. (Prineas, et al., Lab. Invest.38:409-21, 1978; Alvord, J C Koetsier, editor; Handbook of ClinicalNeurol. 3(47):467-502, Koetsier (ed.); Amsterdam; Elsevier SciencePublishers BV, 1985).

Recently, HLA-DR2+transgenic (Tg) mice have been developed that aresusceptible to EAE induced by myelin basic protein (MBP)-85-99 peptide(Madsen et al., Proc. Natl. Acad. Sci. USA 96:10338-43, 1999 (1); Madsenet al., Nat. Genet. 23:343-47, 1999 (2)) and proteolipid protein (PLP)peptide 95-116 (Kawamura et al., J. Clin. Invest. 105:977-84, 2000).Myelin oligodendrocyte glycoprotein (MUG) appears to be recognizedfrequently by T cells from MS patients (KerlerodeRosbo et al., J. Clin.Invest. 92:2602-08, 1993; KerlerodeRosbo et al., J. Autoimmun.11:287-95, 1998), and the MOG-35-55 peptide has been found to be highlyencephalitogenic in rats and mice (Adelmann et al., J. Neuroimmunol.63:17-27, 1995; Mendel et al., Eur. J. Immunol. 25:1951-59, 1995; Johnset al., J. Immunol. 154:5536-41, 1995). It has also been determined thatMOG-specific TCR-Tg mice develop spontaneous autoimmune optic neuritis,another form of autoimmune demyelination (Bettelli et al., J. Exp. Med.197:1073-81, 2003). MOG-35-55 peptide can induce severe chronic EAE inHLA-DRB1*1501-Tg mice (Rich et al., Eur. J. Immunol. 34:1251-61, 2004).A novel single-chain recombinant TCR ligand (RTL) comprised of the α1and β1 domains of DRB1*1501 covalently linked to MOG-35-55 peptide canreverse clinical signs of MOG peptide-induced EAE (Vandenbark et al., J.Immunol. 171:127-33, 2003). Other reports have identified the regionbetween amino acids 85 to 99 of myelin basic protein (MBP) as containingan immunodominant epitope for T cells and autoantibodies in MS brainlesions. The main region of MBP recognized by T cells andautoantibodies, found in MS brain, is reported to be a core motif, HFFK,from MBPp87-99 in patients who are HLA DRB1*1501 DQB1*0602 (HLA DR2)(see, U.S. Pat. No. 6,531,130, issued Mar. 11, 2003). Other MBP peptidesof interest within the invention include a 10-amino acid segment 86-95(VVHFFKNIVT) that also contains the MHC-T cell receptor contact residuesfor T cells recognizing MBP in the context of DRB1*1501 and DQB1*0602.In the epitope center, the residues VHFFK are reported to be importantfor T cell binding and MHC recognition. Recently, the crystal structureof HLA-DR2 with MBP 85-99 was solved, confirming the prediction that K91is the major TCR contact residue, while F90 is a major anchor into thehydrophobic P4 pocket of the MHC molecule.

As described herein, above, the T cell hybrids of the invention can begenerate to exhibit an Ag-specific, TCR-mediated activation response toany cognate Ag of interest, including the foregoing immunodominantepitopes identified in association with MS. By stimulating the T cellhybrids of the invention with these cognate Ags in the context of APCs,an activation response marked by discernable proliferation of the hybridcells is elicited. This activation response, can be specificallyinhibited or enhanced by TCR antagonists or agonists, which provides thebasis for a wide range of assays to screen, identify, design, test andcharacterize novel TCR antagonists or agonists.

In one screening protocol of the invention, The T cell hybrid ischaracterized, as above, by its capacity to exhibit an Ag-specific,TCR-mediated proliferative response following contact with the cognateAg and APC. The T hybrid is cultured under suitable conditions, and at aselected point in culture is exposed to a known or prospective TCRagonist or antagonist (which is a test compound or agent in the assay).

The test compound or agent can, for example, be a RTL as describedherein. RTLs can comprise a MHC II component covalently bound to acognate Ag that is an immunodominant epitope associated with a selectedautoimmune disease, for example MS. The assay can be used to sensitivelymeasure the effect of the RTL on Ag-specific activation of the T cellhybrid, which can be measured by comparing the Ag-stimulatedproliferation response of the hybrid exposed to the RTL before, orafter, Ag-stimulation, and comparing this proliferation to that of thesame T hybrid under similar conditions but not exposed to the RTL.

As described in the examples below, certain RTLs will exert adetectable, inhibitory effect on the Ag-specific proliferative responseof the T hybrid. This effect will be accurately and sensitivelydetectable in the assay methods and compositions of the invention, andmay be paralleled by other activation changes in the T hybrid, forexample by an increase in IL-2 production measured using anIL-2-dependent (e.g., CTLL) cell line. This assay design allows forscreening and optimizing RTLs, including rational design of RTLs havingdirected structural changes in either the MHC component or Ag componentof the RTL

Comparable assays are provided using the T hybrids of the invention toscreen for many different classes of T cell modulatory agents. Thesemodulatory agents may include agents that bind TCRs or otherwiseinterfere with TCR activity. Alternatively, the modulatory agents maybind or interfere with a T cell coreceptor, an MHC molecule of an APC orRTL, or a cognate Ag of a TCR associated with T cell regulation. In oneexemplary assay, the T hybrids are used for screening or rational designof peptide or small molecule mimetics of important structural domains ormotifs of TCRs, MHC molecules, and/or T cell Ags. In the case of a TCRmimetic, for example, a test compound may structurally mimic a portionof a TCR that interacts with Ag, for example an Ag-binding orAg-interactive domain of the TCR. Desired TCR mimetics in this contextwill include peptides having an amino acid sequence that mimics (i.e.,by exact or conservative sequence identity) a functional motif of theTCR and thereby is capable of binding, chelating, sequestering,inactivating, or otherwise impairing TCR-interactions by a TCR ligand orregulatory molecule (e.g., by blocking cognate Ag binding or APCcostimulation) of the TCR. The effect of the test agent in this contextcan be detected by observation of an inhibitory or stimulatory effect onT hybrid proliferation and/or associated TCR-mediated responses in thepresence of the test agent. If the T cell hybrid is contacted with thetest agent, and subsequently or previously subjected to an Ag-specificstimulation (e.g., Ag plus APC, or by exposure to an RTL that activatesor represses the T cell without APCs), the agonistic or antagonisticeffect of the test agent will be indicated by an increase or decrease inproliferation by the hybrid.

Comparable assays are provided that screen, identify and/or characterizetest compounds that structurally mimic a portion of a cognate Ag thatinteracts with the TCR, for example a TCR-binding or other interactivedomain of the peptide. Desired peptide mimetics in this context, forexample, will include peptides having an amino acid sequence that isaltered from a native sequence (i.e., a naturally occurring, wild-typeor mutant sequence) of a T cell epitope, most often a T cell epitopeassociated with an immune disorder. The test peptide may thereby exhibitmodified TCR-binding and/or TCR-activation kinetics, which can bedetected by observing inhibitory or stimulatory effects on T hybridproliferation (and/or IL-2 production, etc.). If the T cell hybrid iscontacted with the test peptide agent, and subsequently or previouslysubjected to an Ag-specific stimulation (e.g., Ag plus APC, or byexposure to an RTL that activates or represses the T cell without APCs),the agonistic or antagonistic effect of the test peptide will correlatewith an increase or decrease in proliferation by the hybrid.

Various other assays are contemplated that incorporate the novel Thybrids of the invention in known assay formats. These include highthroughput assays designed to detect modulators of T cell activity, TCRactivity, MHC activity, and Ag effects on T cell biology, among othertargets.

The T hybrids and assays of the invention are useful in developingdiagnostic and therapeutic methods and compositions for MS, and also fora variety of other autoimmune disorders. Key targets for constructingthe hybrids and designing the subject assays include TCRs, MHCmolecules, and cognate Ags associates with the subject immune disorder.Another important autoimmune disease in this context is rheumatoidarthritis (RA). RA is an autoimmune disease that primarily affectsperipheral joints with cartilage destruction and subsequent boneerosion. In a significant fraction of patients, this persistentsynovitis leads to destruction of articular cartilage and surroundingstructures and is a cause of significant morbidity. Importantly, themajor genetic contribution to RA involves HLA class II alleles dominatedby HLA-DRB1*0401 and *0404 (in DR4) and DRB1*0101 (in DR1) in Caucasianpopulations. These alleles all share a sequence motif at positions 67-74of the third hypervariable region of the DRβ chain, termed the sharedepitope (Gregerson et al., Arthritis Rheum. 30:1205-13, 1987), whichprofoundly affects peptide binding and CD4+T cell recognition (Nepom,Adv. Immunol. 68:315-32, 1998). Synovial tissue and fluid from inflamedjoints of RA patients usually contain large numbers of CD4+ T cells,including activated CD4+ T cells. Studies of synovial T cells also havedemonstrated sets of related oligoclonal CD4⁺ T cell expansions inindividual patients that express highly homologous TCRs (Striegich etal., J. Immunol. 161:4428-36, 1998). Based on these findings, it hasbeen hypothesized that disease-associated HLA-DR molecules presentarthritogenic cartilage antigens and cause stimulation and expansion ofantigen-specific T cells in the joint. This T cell response then drivesthe inflammatory process.

Previous studies have suggested that type II collagen (CII) and humancartilage gp39 (HCgp39) are among the most likely synovial antigens tobe involved in T cell stimulation in RA. CII, the main constituent ofhyaline cartilage, has been proposed as an important autoantigen in RAbecause CII-specific antibodies are frequently found in RA patients andbecause an RA-like disease can be induced in certain murine strainsafter immunization with CII. Studies performed in DR4- andDR1-expressing mice have located an immunodominant T cell epitope toposition 263-270 in CII by using synthetic peptides (Fugger et al., Eur.J. Immunol. 26:928-33, 1996; Rosloniec et al., J. Immunol. 160:2573-78,1998; Andersson et al., Proc. Natl. Acad. Sci. USA 95:7574-79, 1998). Inlater studies, Backlund and coworkers (Proc. Nat. Acad. Sci. USA99:9960-65, 2002) used a humanized mouse model expressingHLA-DRB1*0401/DRA1*0101, human CD4, and human CII (huCII) on abackground deficient of murine class II expression (Fugger et al., Proc.Natl. Acad. Sci. USA 91:6151-55, 1994; Malmstrom et al., Scand. J.Immunol. 45:670 77, 1997) to further elucidate the role and behavior ofT cells in RA. From these studies the authors reported a dominant T cellresponse to glycosylated CII-glycopeptides in a cohort of severelyaffected RA-patients.

Further experiments have defined dominant peptide determinants of CIIand gp39 antigens when presented by HLA-DR4, the most importantRA-associated HLA type. Kotzin et al., (Proc. Nat. Acad. Sci. USA97:291-96, 2000) used fluorescent, soluble peptide-DR4 complexes(tetramers) to detect synovial CD4+T cells reactive with CII and HCgp39in DR4+ patients. The CII-DR4 complex bound in a specific manner to CIIpeptide-reactive T cell hybrids, but did not stain a detectable fractionof synovial CD4+ cells. A background percentage of positive cells(<0.2%) was not greater in DR4 (DRB1*0401) patients compared with thosewithout this disease-associated allele. Similar results were obtainedwith the gp39-DR4 complex for nearly all RA patients. In a small subsetof DR4+ patients, however, the percentage of synovial CD4+ cells bindingthis complex was above background and could not be attributed tononspecific binding. These studies demonstrate the potential forpeptide-MHC class II tetramers to be used to track antigen-specific Tcells in human autoimmune diseases. Together, the results also suggestthat the major oligoclonal CD4+ T cell expansions present in RA jointsare not specific for the dominant CII and HC 39 determinants.

The T hybrids and related assays and compositions of the invention arealso adapted for scientific and clinical investigation and management oftype 1 diabetes, or IDDM. IDDM results from the destruction ofpancreatic islet β cells by a complex autoimmune process to which bothgenetic and environmental factors appear to contribute. In humans, as inthe accepted model for the human disease, NOD mice, the main geneticcontribution to susceptibility resides in class II loci of the MHC, withparticular sequences and structures in haplotypes that confersusceptibility in NOD mice and in diabetic human patients (Hattori etal., Science 231:733-35, 1986; Acha-Orbea et al., Proc. Natl. Acad. Sci.USA 84:2435 39, 1987; Corper et al., Science 288:505-11, 2000; Latek etal., Immunity 12:699 710, 2000; and Lee et al., Nat. Immunol. 2:501-07,2001). In particular, IDDM is genetically associated with specificalleles from the HLA-DQ and HLA-DR loci shown to contribute to thedisease. Among DR4 subtypes, HLA-DRB1*0401, HLA-DRB1*0402, andHLA-DRB1*0405 alleles lend susceptibility, while HLA-DRB1*0403 confersprotection. In addition, glutamic acid decarboxylase isoform 2 (GAD65)has been identified as a key target autoantigen of IDDM.

IDDM is marked by the presence and activation of autoreactive T cells.Transgenic models have shown that, for the most part, T cells reactiveagainst peripheral antigens are selected in the thymus much like other Tcells are (Katz et al., Cell 74:1089-100, 1993; Lafaille et al., Cell78:399-408, 1994; Goverman et al., Cell 72:551-60, 1993; and Verdagueret al., J. Exp. Med. 186:1663-76, 1997).

Several autoreactive T cell clones isolated from diabetic NOD mice havebeen shown to be pathogenic in transfer experiments (Pankewycz et al.,Eur. J. Immunol. 21:873-79, 1991; Daniel et al., Eur. J. Immunol.25:1056-62, 1995); Haskins et al., Science 249:1433-36, 1990). BDC-2.5is the best characterized of these clones, and has been shown to greatlyaccelerate disease when transferred to young animals. This CD4+ Th1clone displays a Vβ4/Vα1 (AV1S5) TCR heterodimer, and is restricted byAg7, the lone MHC class II molecule of NOD mice (Candeias et al., Proc.Natl. Acad. Sci. USA 88:6167-70, 1991; Acha Orbea et al., Proc. Natl.Acad. Sci. USA 84:2435-39, 1987). The nature of the presented antigen isstill unknown, although it has been shown to be associated with themembrane fraction of β granules (Bergman et al., Diabetes 43:197-203,1994). Mice transgenic for the BDC-2.5 TCR show a robust positiveselection of CD4+ cells in the thymus. These are then exported, naiveand fully reactive, to peripheral lymphoid organs and later infiltratethe pancreatic islets in a precocious and synchronized manner (Katz etal., Cell 74:1089-100, 1993).

As noted above, Patel and coworkers (Proc. Natl. Acad. Sci. USA94:8082-87, 1997) utilized HLA-DR (DR0401), human CD4, murine class IInull triple transgenic mice and recombinant GAD65 to generate T cellhybrids, and made overlapping sets of peptides to map the immunodominantepitopes of the GAD65 autoantigen. These authors identified 10immunogenic regions for GAD65. These epitopes were also reportedlygenerated by human antigen-presenting cells, and their presentation isDR0401 restricted (as shown by the use of typed human lymphoblastoidcell lines and antibody blocking experiments). Immunodominant GAD65epitopes defined in transgenic mice correspond to GAD65 regionspreviously shown to elicit T cell responses specifically in DR0401 IDDMpatients. Although the major epitopes contain DR0401 binding motifs, oneof the epitopes contains a DR0405 motif Additional immunogenic epitopesfor use within the invention have been identified by Wicker et al., (J.Clin Invest. 98:2597-603, 1997), Lohmann et al., (Lancet 343:1607-08,1994), and Lohmann et al., (J. Autoimmun. 9:385-89, 1996).

From these studies, exemplary IDDM-associated candidate peptides for usein generating hybrids and related compositions and methods within theinvention include, but are not limited to: LYNIIKNREG; YNIIKNREG;LYNIIKNRE; LIAFTSEHS; FTSEHSHFS; FFRMVISNPAA; FRMVISNPA; andSLRTLEDNEER.

The T hybrids and related assays and compositions of the invention arealso adapted for scientific and clinical investigation and management ofpsoriasis and other skin diseases known to exhibit autoimmune etiology.Onset of psoriasis may be triggered by systemic infections such as strepthroat, skin injury, vaccinations, and certain oral medications such assteroids. Subsequently, the immune system is thought to induceinflammation and excessive skin cell reproduction, which can beexacerbated by additional factors such as stress and diet.

Previous reports have described a monoclonal antibody designated UM4D4which recognizes the cell surface marker CDw60. This marker is presenton a subset of normal T cells, melanocytes, malignant melanoma cells,and hyperproliferative psoriatic keratinocytes. CDw60 antibodies bind tothe acetylated form of GD3. 74% of basal cell carcinomas express CDw60,whereas CDw50 expression in normal skin is confined to melanocytes and afew scattered keratinocytes at the basal cell layer. Psoriatic skin,basal and suprabasal keratinocytes all express CDw60. Cloned T celllines obtained from lesional skin upon initiation have been shown torelease soluble factors including IL-4 and IL-13, that up-regulatedCDw60 expression on cultured normal keratinocytes. Consequently, CDw60and other markers associated with psoriasis and other immune disordersof the skin also serves as targets and test agents within the methodsand compositions of the invention.

The T hybrids and related assays and compositions of the invention arealso adapted for scientific and clinical investigation and management oftransplant complications, including graft-versus-host-disease (GVHD).Specific suppression of a host's immune response to donor HLA antigensremains the ultimate goal for clinical transplantation. In spite ofconsiderable effort, however, allospecific human suppressor T cells (Ts)have been difficult to generate. Previous studies have shown thatallospecific and xenospecific T suppressor cells can be raised bymultiple priming of T cells in mixed lymphocyte cultures (MLC).Allospecific T suppressor cells prevent the upregulation of B7 moleculeson target APCs, interfering with the CD28-B7 interaction required for Thelper cell activation.

Transplant tolerance has been induced in adult animals by inactivationor depletion of mature T lymphocytes prior to transplantation usingcyclosporine (CsA), total lymphoid irradiation, anti-lymphocyte serum,antibodies against CD4+ and CD8+ T cells, or donor-specifictransfusions. Studies of peripheral graft tolerance have suggested theexistence of an active mechanism of suppression which is donor-specificand can be transferred adoptively to secondary hosts. However, there isstill controversy concerning the phenotypic characteristics of theseregulatory T cells and their MHC restriction, as both CD8+ and CD4+ Tcells have been reported to display suppressive activity. It has beensuggested that suppression may result from antagonistic effects of(Th)2-type lymphokines (such as IL-4 and IL-10) on the response of Thelper cells, or from recognition by T suppressor cells of eitheridiotypic determinants of the TCR of alloreactive T cells or of MHCantigens expressed on stimulating cells. The generation of T suppressorlines has proven, however, to be a difficult task rendering thecharacterization of these cells hard to achieve.

Xenospecific T suppressor cells prevent the up-regulation of CD154molecules on the membrane of T helper (Th) cells, inhibiting theirability to react against the immunizing MHC-class II xenoantigens. Themechanism of this suppression, therefore, appears to be blockade ofCD154/CD40 interaction required for efficient costimulation of activatedT cells. Additional studies have shown that T suppressor cell lines canbe generated by in vitro immunization of human PBMCs with syntheticpeptides or soluble proteins coupled to beads. Such CD8+ T suppressorcells exhibit antigen specificity and restriction by self MHC class Imolecules, limited TCR V beta gene usage, ability to inhibitantigen-specific, MHC Class II restricted, T helper cell proliferativeresponses, and capacity to downregulate and/or inhibit the upregulationby T helper cells of CD40, CD80, and CD86 molecules on APCs. Thesefindings provide a basis for the development of specificimmunosuppressive tools and therapies targeting regulation of immuneresponses in graft rejection using the T hybrid cells and assay methodsof the invention as described herein.

RTLs and other compositions provided or identified using thecompositions and methods of the invention are useful to treatindividuals suffering from immune disorders, including demyelinatingautoimmune diseases. Diagnosis of MS patients may utilize a variety ofcriteria known to those of skill in the art. A quantitative increase inmyelin autoreactive T cells with the capacity to secrete IFN-γ isassociated with the pathogenesis of MS and EAE. During thepresymptomatic period there is infiltration of leukocytes into thecerebrospinal fluid, inflammation and demyelination. Family historiesand the presence of the HLA haplotype DRB1*1501, DQA1*0102, DQB1*0602are indicative of a susceptibility to the disease. Treatment during theearly stages of the disease is preferred, in order to slow down orarrest the further loss of neural function.

Patients are diagnosed as having MS according to conventional clinicalcriteria. Such criteria rely on the presence of two attacks at least onemonth apart, where an attack is a sudden appearance of or worsening ofan MS symptom or symptoms which lasts at least 24 hours; and more thanone area of damage to central nervous system myelin. The damage tomyelin must have occurred at more than one point in time and not havebeen caused by any other disease that can cause demyelination or similarneurologic symptoms.

MRI (magnetic resonance imaging) is the preferred method of imaging thebrain to detect the presence of plaques or scarring caused by MS,although CT scans may also be used. Other symptoms include disability inmental, emotional, and language functions, movement and coordination,vision, balance, and the functions of the five senses. Evoked potentialtests are electrical diagnostic studies which can show if there is aslowing of messages in the various parts of the brain, and may provideevidence of scarring along nerve pathways that is not apparent on aneurologic exam. Cerebrospinal fluid, usually taken by a spinal tap, maybe tested for levels of cytokines, and for the presence of oligoclonalantibody band.

The therapeutic effect of compositions provided or identified byimplementation of this disclosure may be measured in terms of clinicaloutcome, or may rely on immunological or biochemical tests. Suppressionof the deleterious T cell activity can be measured by enumeration ofmyelin-reactive Th1 cells in spinal fluid, by quantitating the releaseof cytokines at the sites of lesions, or using other assays for thepresence of autoimmune T cells known in the art. Alternatively, one maylook for a reduction in symptoms of a disease, such as the damage toneural tissue observed in MS, or the decrease in-the number or severityof attacks of MS suffered by MS patients. Damage to neural tissue can beassessed for example by magnetic resonance imaging (MRI) and measurementof the number and severity of lesions visible therein. Reduction in MSattack number or severity can be assessed for example by clinicalevaluation of patients. Methods for both MRI and clinical evaluation arewell-known in the art.

Various methods for administration immune modulatory agents of theinvention may be employed. The agents may be administered orally orinjected, e.g. by intravascular, intratumor, subcutaneous,intraperitoneal, intramuscular, injection. The dosage of therapeuticformulations comprising the immune modulatory agents will vary widely,depending upon the nature of the disease, the frequency ofadministration, the manner of administration, the clearance of the agentfrom the host, and the like. The initial dose may be larger, followed bysmaller maintenance doses. The dose may be administered as infrequentlyas weekly or biweekly, or fractionated into smaller doses andadministered daily, semi-weekly, etc. to maintain an effective dosagelevel. In many cases, oral administration will require a higher dosethan if administered intravenously.

Therapeutic compositions of the invention may also contain othertherapeutically active agents, e.g. immunosuppressants, β-interferon,steroids, etc. Of particular interest are combinations with other agentscapable of additive or synergistic effect in achieving a therapeuticresult, e.g. where a different or complementary pathway is affected byeach of the active agents. Immunosuppressants of interest includecyclosporins A and G, FK-506, mycophenylate mofetil, rapamycin,azathioprine, antibodies for plasma membrane proteins associated withgraft rejection, such as antibodies to CD4, CD8, CD2, LFA-1, ICAM-1,CD28, and the like; and immunosuppressive oligopeptides derived from MHCmolecules. Antibacterial, antiviral and antifungal drugs may also beco-formulated in order to minimize the effects of immunosuppression.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the specificpeptides are more potent than others. Preferred dosages for a givenimmune modulatory agent are readily determinable by those of skill inthe art according to the teachings herein supplemented by knowledge inthe art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the methodsand methodologies that are described in the publications which might beused in connection with the presently described invention. Thepublications discussed above and throughout the text are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior invention.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g., amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for.

Examples

Within the following examples, BW5147 variant cells were used as fusionpartners to generated CD4+ T cell hybrids that strongly respond to bothhuman and mouse MOG-35-55 peptides and uniquely express TCR BV8 inconjunction with AV17 and AV11 for the recognition of human MOG-35-55peptide restricted by the HLA-DR2/DRB1*1501 allele. Additionally, theseexamples show that MOG peptide-specific proliferation and cytokineresponses of these T cell hybrids can be inhibited by a specific RTLcontaining HLA-DR2 external domains covalently linked to the cognatehuman MOG-35-55 peptide. This reduction of activation of the T cellhybrids in vitro by the RTL was reflected by the RTL's ability toinhibit EAE induced by human MOG-35-55 in DR2-Tg mice. These datademonstrate the powerful utility of peptide-specific T hybrid cells as astandardized biological screening tool.

DR2 Transgenic Mice

HLA-DR2 (DRB1*1501 and DRB1*1502) mice used herein have been describedpreviously (Rich et al., Eur. J. Immunol. 34:1251-61, 2004; Gonzalez-Gayet al., Hum. Immunol. 50:54-60, 1996). The mice were bred and housed atthe Portland Veterans Affairs Medical Center, Portland, Oreg., underpathogen-free conditions according to institutional guidelines.Offspring were screened by flow cytometry by examining transgeneexpression on APC from whole blood obtained from the tail.

Antigens

Mouse (m)MOG-35-55 peptide (MEVGWYRSPFSRVVHLYRNGK), human (h)MOG-35-55peptide (MEVGWYRPPFSRVVHLYRNGK), MBP-85-99 peptide and mouse PLP-95-116peptide were synthesized using solid-phase techniques and purified byHPLC at the Beckman Institute, Stanford University (Palo Alto, Calif.).Purified protein derivative (PPD) was purchased from the StatenInstitute (Copenhagen, Denmark). Con A was purchased from Sigma-Aldrich(St. Louis, Mo.).

MOG-35-55-Specific T Cell Lines

Tg mice were immunized with mMOG-35-55 or hMOG-35-55 peptides in CFAcontaining heat-killed Mycobacterium tuberculosis, strain H37RA (Difco,Detroit, Mich.). T cells were recovered from the spleen or draininglymph nodes of the Tg mice 10 days after immunization and cultured ascell lines in 2% fetal bovine serum (FBS)-containing RPMI 1640 mediumsupplemented with 0.05 mM 2-mercaptoethanol, 2 mM L-glutamine, 1 mMsodium pyruvate, 100 μg/ml penicillin G, 100 μg/ml streptomycin (GIBCO,New York) and 5 ng/ml r1L-2 (R & D Systems, Minneapolis, Minn.). Thesecell lines were re-stimulated with 10 μg/ml MOG-35-55 peptide in thepresence of irradiated (2500 rad) syngeneic antigen-presenting cells(APC) for 2 days followed by culturing in rIL-2-containing medium foradditional 3 days prior to cell fusion. Proliferation responses wereassessed by using 4×10⁴ T cells plus 2×10⁵ syngeneic APC per well with1% FBS-containing stimulation medium alone or in the presence of Ags atvaried concentrations in triplicate. Cultures were incubated for 72 hrat 37 C in 7% CO₂ and pulsed for the final 18 hr with 0.5 μCi per well[³H]thymidine (Amersham, Arlington Heights, Ill.).

BW5147 Tumor Cell Lines

AKR BW5147.G.1.4 cell line was purchased from the American Type CultureCollection (ATCC, Cat. #TIB 48) and cultured in 10% FBS-containing RPMI1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate,100 μg/ml penicillin G, 100 μg/ml streptomycin, and 0.1 mM non-essentialamino acids (Invitrogen, Carlsbad, Calif.). The BW5147 variant cell linewas obtained from the National Jewish Medical and Research Center,Denver, Colo. and cultured in the same medium as that used forBW5147.G.1.4 cell line.

Cell Fusion and T Cell Hybrid Selection

Cell fusion was accomplished by using 50% polyethylene glycol 1500 (PEG1500) (Roche Diagnostics, Mannheim, Germany) with procedures asrecommended by the manufacturer. After fusion, the cells were culturedin selection medium, e.g. 10% FBS-containing RPMI 1640 culture mediumsupplemented with 1× hypoxanthine aminopterin thymidine (HAT)-mediasupplement (GIBCO), and distributed as 0.2 ml per well in a 96-wellplate. After 2 weeks, unfused T cells gradually died off because of thelack of exogenous IL-2, and unfused HAT-sensitive BW tumor cells alsodied off, leaving only the dividing HAT-resistant T cell hybrids. Thybrid cells from each well were removed respectively to individual T25tissue culture flasks (Costar, Corning, N.Y.) containing HAT-free mediumsupplemented with “Tumor Cocktail Star” supplied by the National JewishMedical and Research Center (Denver, Colo.).

Characterization of T Cell Hybrid

IL-2 bioassay: 10⁵ hybrid cells per well of a 96-well plate in 100 μl of1% FBS-containing medium were combined with 10⁶ irradiated (2500 rad)syngeneic APC or irradiated (4500 rad) 4×10⁴ HLA-DRB1*1501-transfected 1cells (Klohe et al., 1988)(a gift of Dr. R. Karr, Searle Research andDevelopment, St. Louis, Mo.) in 100 μl alone or in the presence of 10μg/ml of individual peptides or 5 μg/ml Con A, and incubated at 37 C and7% CO₂ for 24 hr. Supernatants were collected from the top of theculture, followed by centrifugation for 1 min at 1000 rpm. Hybridsupernatants were added in triplicate at 50, 25, 10, or 2% into wellscontaining 5,000 CTLL-2 cells in 100 μl of 1% FBS culture medium. After24 hr of culture, the cells were pulsed with 0.5 μCi [³H]Thymidine foran additional 6 hr. Stimulation index (SI) was calculated as the cpm ofAg-stimulated cultures divided by the cpm of the cells cultured inmedium.

FACS analysis: Hybrids and BW tumor cells were stained with 4 μl of thefollowing antibodies: anti-CD3-FITC, anti-CD4-PE, anti-CD8-FITC, andanti-mouse IgG1-PE (BD PharMingen, Calif.) for 30 min at 4 C. Afterwashing 2×, the cells were analyzed using Cell Quest™ software on aFACScan. Hybrids were stimulated with 5 μg/ml Con A for 2 days in thepresence of DRB1*1501-transfected L cells, and Brefeldin A was added forthe last 6 hr followed by cell surface staining with anti-CD4-FITC. Allsurface-stained cells were washed 2× with p17.2 PBS, then fixed andpermeabilized as defined by the manufacturer (BD PharMingen).Permeabilized cells were stained respectively with anti-IL-2-PB,anti-IFN-γ-PE and anti-TNF-α-PE. Anti:mouse TCR beta-Cy-Chrome andanti-AV11-FITC have been used for cell surface staining andintracellular staining.

RT-PCR Analysis of TCR V Gene Expression

Relative AV and BV gene expression was analyzed by semi-quantitative PCRas previously described (Buenafe et al., J. Neurosci. Res. 47:489-99,1997) with modifications. Briefly, cDNA was synthesized from total RNAwith oligo(dT) and Superscript II reverse transcriptase (LifeTechnologies, Gaithersburg, Md.). BV-specific PCR products weregenerated using BV-specific primers and a fluorescent Tet-labeled BCprimer with an application profile: 26 cycles at 94.5 C for 30 s, 55 Cfor 60 s, and 72 C for 60 s followed by a 5-min extension time at 72 C.AV-specific PCR products were similarly generated using AV primers and afluorescent Cy3-labeled AC primer, mCab (Casanova et al., J. Exp. Med.174:1371-83, 1991), using the same amplification profile. PCR productswere separated on a polyacrylamide gel and the gels were scanned for thepresence of fluorescent bands using a Bio-Rad Molecular Imager FX(Bio-Rad Laboratories, Hercules, Calif.).

RTL Preparations

Single chain human RTLs containing approximately 200 amino acid residuesderived from HLA-DR2b (DB1*1501) have been described previously (Changet al., J. Bio. Chemistry 276:24170-76, 2001; Huan et al., J. Chem.Technol. & Biotechnol. in press, 2004). These RTLs were designed usingthe same principles as for rat RTLs and have been produced inEscherichia coli with and without amino-terminal extensions containingantigenic peptides as described previously (Burrows et al., Prot. Eng.12:771-78, 1999; Chang et al., J. Bio. Chemistry 276:24170-76, 2001).Structural characterization using circular dichroism predicted thatthese molecules retained the antiparallel β-sheet platform andantiparallel α-helices observed in the native HLA-DR2 heterodimer.DR2-derived RTLs exhibited a cooperative two-state thermal unfoldingtransitions. When covalently linked to human MOG-35-55 or MBP-85-99peptides, the RTLs showed increased stability to thermal unfoldingrelative to empty DR2-derived RTLs. To modify the β-sheet platform ofthe DR2-derived RTLs that formed higher order aggregations, asite-directed mutagenesis was performed to obtain a monomeric form ofRTL which contained covalently tethered antigen peptides and capable ofexhibiting its biological functions (Huan et al., J. Chem. Technol. &Biotechnol. in press, 2004). RTL VG1000 is a DR2-derived monomeric RTLcovalently linked to human MOG-35-55 peptide (Virogenomics, Portland,Oreg.). RTL340 is also a DR2-derived monomeric RTL, but covalentlylinked to MBP-85-99 peptide produced in our laboratory. Both RTL VG 1000and RTL340 are dissolved in 10 mM Tris buffer stock solution andaliquotted at a concentration of 40 uM and stored at −80 C.

RTL In Vitro Potency Assay Using T Cell Hybrids

Human MOG-35-55 peptide-specific T cell hybrid cells (2×10⁵/well) wereco-cultured in triplicate with 2 mM Tris-containing medium alone, 8 uMRTL VG1000 or 8 uM RTL340 in 2 mM Tris-containing medium respectivelyfor 24, 48, 72 and 96 hr with uptake of [³H]Thymidine assessed for thelast 6 hr before the end of each culturing time. The culture plates wereharvested and the proliferation levels of cultured hybrid cells werecalculated and compared by net cpm (mean±SD). Student's t test was usedto assess significant differences (p<0.05). Aliquotted hybrid cellcultures were thoroughly washed with RPMI after 24 hr and 96 hrco-culturing and further stimulated with and without 10 μg/ml hMOG-35-55peptide presented by irradiated (4500 rad) DRB1*1501-transfected celllines at a 1:1 ratio in triplicate for 48 hr. Half of the supernatantwas collected from top of each well and transferred into correspondingwells of another culture plate in which 100 μl of 1% FBS-containingmedium with 5000 CTLL cells per well had been seeded. After 24 hr ofculture, the CTLL cells were pulsed with [³H]Thymidine for an additional6 hr. The net cpm (mean±SD) were calculated and compared betweendifferent conditions.

Induction of Active EAE and Treatment with RTL VG1000

Transgenic HLA-DR2 male and female mice between 8 and 12 weeks of agewere immunized subcutaneously at four sites on the flanks with 0.2 ml ofan emulsion comprised of 200 μg human MOG-35-55 peptide in CFAcontaining 400 μg heat-killed Mycobacterium tuberculosis H37RA (Difco,Detroit, Mich.) as described previously (Vandenbark et al., J. Immunol.171:127-33, 2003). In addition, mice were given pertussis toxin (Ptx,List Biological Laboratories, Campbell, Calif.) on Days 0 and Day 2post-immunization (75 ng and 200 ng per mouse, respectively). Mice weretreated i.v. daily for 8 days, beginning on the second day after onsetof clinical signs, with 100 μl of 1 mg/ml RTL VG1000 or vehicle (Tris,pH 8.5). Actively immunized mice were assessed daily for clinical signsof EAE according to the following scale: 0=normal; 1=limp tailor mildhindlimb weakness; 2=limp tail and moderate hindlimb weakness or mildataxia; 3=limp tail and moderately severe hindlimb weakness; 4=limp tailand severe hindlimb weakness or mild forelimb weakness or moderateataxia; 5=limp tail and paraplegia with no more than moderate forelimbweakness; and 6=limp tail and paraplegia with severe forelimb weaknessor severe ataxia or moribund condition. The mean disease score andstandard deviation were calculated on each day for the affected mice.

For generating MOG-35-55-specific T cell lines used for fusion,splenocytes or lymphocytes from draining from draining lymph nodes ofHLA-DRB1*1501-Tg mice immunized with either mMOG-35-55 or hMOG-35-55peptides were tested for Ag-specific responses. Two T cell linesresponded strongly to either mMOG-35-55 or hMOG-35-55 peptide that wasused initially for immunization, as shown in part A and B of FIG. 1. A Tcell line selected from mMOG-35-55 peptide-immunized spleen cells hadthe same level of cross-recognition of hMOG-35-55 peptide at aconcentration 10 μg/ml, but 3-fold lower responsiveness to hMOG-35-55peptide at lower concentrations of 2 and 0.4 μg/ml, as is shown in FIG.1C. This result suggests that a high functional TCR avidity wasdeveloped in recognition of the immunizing peptide antigen versus thecross-reactive peptide (Margulies, D. H., 2001). The humanMOG-35-55-reactive lines were also cross-reactive to mouse MOG-35-55peptide at the same responsive levels with the same Ag dose-dependentpattern.

AKR BW5147.G.1.4 thymoma cells are resistant to 6-thioguanine and die inhypoxanthine aminopterin thymidine (HAT)-selective medium (Goldsby etal., Nature (London) 267:707-08, 1977). This fusion partner did notrespond to MOG-35-55 peptides or PPD, and its response to Con A wasslightly inhibited, consistent with previous reports (Indrova et al.,Folia. Biol. (Praha) 30:390-95, 1984). Although BW5147 does not expresssurface TCR (Yague et al., Cell 42:81-87, 1985), isolation of cDNAclones from hybrids indicated that BW5147 had at least one functionalα-chain and one functional β-chain rearrangement (Barth et al., Nature316:517-23, 1985). BW5147 expressed BV1, BV5 and AV1, AV11 and AV16S1P(BW.B)(Letourneur et al., Eur. J. Immunol. 19:2269-74, 1989), as shownin FIG. 2 (top panel), which is consistent with previous reports (Chienet al., Nature 312:31-35, 1984; Blackman et al., Cell 47:349-57, 1986;Barth et al., Nature 316:517-23, 1985). BW5147 variant cells are alsotruly no longer expressing BV1, AV1 and AV11 as shown in FIG. 2 (thirdpanel). This result is consistent with that originally demonstrated byan RNA dot blot hybridization analysis (White et al., J. Immunol.143:1822-25, 1989). However, the BV5 and AV16S1P (BW.B) products werestill present in these BW5147 variant cells. Neither BW5147 nor itsvariant were found to express TCR on the cell surface, as we confirmedby negative staining data shown in FIG. 3A. However, endogenousexpression of TCR V genes by BW5147 was detected by positiveintracellular staining of 37% of cells using anti-TCR beta-Cy-Chrome(FIG. 3B), whereas the B8W5147 variant had essentially no intracellularstaining for TCR •• chains.

The TCR repertoire used in response to MOG peptides was next determined.According to prior reports, after MOG peptides were injected intoC57BL/6J mice, the mice developed significant T cell responses to MOGpeptides 1-21, 35-55 and 104-117. However, MOG-35-55 was the only MOGpeptide that induced neurological impairment in C57BL/6J mice (Mendel etal., Eur. J. Immunol. 25:1951-59, 1995). The encephalitogenicMOG-35-55-reactive T cell lines predominantly expressed the BV8 geneproduct (40-43%) (Mendel et al., Eur. J. Immunol. 25:1951-59, 1995). Thepresently disclosed MOG-35-55-reactive T cell hybrids, H2-1 and H6-1,uniquely expressed BV8 as shown in FIG. 2 (Panel 2 and 4). Theutilization of AV gene products by hybrid clones was also examined,which revealed that the clone H2-1, which was an hMOG-35-55-selected Tcell fused with a BW5147 variant cell, expressed AV17S3 (BM.B) (Couez etal., Int. Immunol. 3:719-29, 1991; Ballenthin et al., J. Neurosci. Res.46:271-81, 1996) and AV11 as shown in FIG. 2 (Panel 4). Clone H6-1, amMOG-35-55-selected T cell fused with a BW5147 cell, expressed AV1, AV11and AV16S1P (BW.B) as shown in FIG. 2, Panel 2. Because of theavailability of anti-AV11-FITC (RR8-1) (BD PharMingen), the clones H2-1and H6-1 and the BW5147 cells were stained with this Ab, which revealeda clear positive expression of AV11 on the cell surface of H2-1, but nodetectable staining for AV11 on H6-1 or BW5147, as shown in FIG. 3C.These data suggest that AV11 is used for MOG-35-55-recognition by cloneH2-1. However, AV11 in clone H6-1 is only expressed endogenously,apparently adopted from the fusion partner BW5147, and the significantAV gene used for Ag-recognition could not be otherwise identified. Infuture studies, we will evaluate additional AV gene primers by RT-PCR,as well as mAbs for other AV genes.

Interestingly, both clone H2-1 initially selected with hMOG-35-55 andclone H6-1 initially selected with mMOG-35-55 used BV8 in combinationwith a certain set of AV genes, in spite of the one amino aciddifference at position 42 of the two MOG-35-55 sequences. Thisbiological phenomenon may be due to the fact that this amino acid change(serine residue 42 of mouse MOG-35-55 changed to proline in the humansequence) does not dramatically alter interaction of these peptides tothe DRB1*1501 binding pocket (Ballenthin et al., J. Neurosci. Res.46:271-81, 1996), thus minimizing conformational changes that mightaffect selection of TCR V genes.

Phenotypically, T cell hybrids expressed CD3 and CD4 at levels greaterthan 90%, as is shown in FIG. 4A. The cytokine expression pattern showedthat the CD4+ T cell hybrid clones had strongly enhanced expression ofIL-2 (30%), TNF-α (44%), and IFN-γ (24%), but not IL-4 or IL-10, uponCon A stimulation for 48 hr (FIG. 4B). This result indicates that theseT cell hybrid cells are Th1 type. Although BW5147 cells can suppress Th2cytokine production (Weiss et al., 1997) and may influence the cytokinepattern of the hybrids, it is also evident that the Th1 type patternexpressed by hybrid clones is representative for MOG-35-55-specific Tcells selected from the immunized mice, because the MOG-35-55-specific Tcells used for the fusion showed a similar cytokine profile.

Functionally, clone H2-1 strongly responded to hMOG-35-55 peptide byproliferation in the presence of DR81*1501-transfected APC (directassay) (SI 8×) and an indirect assay of culture supernatants on CTLL-2cells (SI 18×), as shown in FIG. 5A. The hybrids H6 and AS, bothgenerated from fusions between mMOG-35-55-specific T cells and BW5147cells, showed strong responses to mMOG-35-55 and Con A (FIG. 58).Regarding cross-reactivity, the 1-12 hybrid was strongly cross-reactivewith mMOG-35-55 (SI 10×) and the H6 responded equally well to bothMOG-35-55 peptides at 10 μg/ml. The A5 was also cross-reactive withhuman MOG-35-55 peptide as is shown in FIG. 5C. Finally, the H2 hybridwas found not to respond to M8P-85-99, PLP-95-116, or PPO (FIG. 5D).

To identify MHC restriction molecules, three types of APCs were used topresent Ag to the H2 hybrid. These APC were 1) syngeneic irradiatedthymocytes with no expression of mouse MHC class II molecules but a 40%expression of HLA-DR; 2) DR81*1501-transfected L cells; and 3)DR85*0101-transfected L cells (Klohe et al., J. Immunol. 141:2158-63,1988). As shown in FIG. 5E, the H2 hybrid responded best to MOG-35-55peptides and Con A by using syngeneic APC. The DR81*1501-transfected APCwere able to stimulate T cell responses to Ag and Con A up to 75% of thesyngeneic APC levels. However, DR85*0101-transfected APC were not ableto stimulate any Ag-specific T cell responses, but stimulated Con Aresponse. These data clearly demonstrate DR81*1501 restriction of theMOG-35-55-stimulated T cell responses, consistent with the result fromthe systematic study of mapping T cell epitopes of MOG in HLA Tg mice byChella David's laboratory (Khare et al., Int. Immunol. 15:535-46, 2003).The hybrid clone was stimulated with MOG-35-55, generating a cytokine(Th1) profile very similar to that induced by Con A stimulation (datanot shown).

Having demonstrated specificity and MHC restriction, furtherinvestigations were conducted on the humanized T cell hybrids fortesting inhibitory signaling properties of novel recombinant TCR ligands(RTLs), which have the capacity to inhibit EAE in vivo. In particular,an in vitro test was devised to assess the biological activity of VG1000(external domains of DR2 linked to hMOG-35-55 peptide) that is acandidate for human therapy trials in MS. As is shown in FIG. 6A,constitutive proliferation responses of H2-1 hybrid cells co-culturedwith RTL VG1000 for 24, 48, 72 or 96 hr all were significantly inhibitedcompared to the same cells co-cultured with medium containing identicalamount of Tris buffer or RTL340 containing the same DR2 moiety butcovalently linked to M8P-85-99 peptide. In a second type of assay,aliquots of H2-1 hybrid cells were co-cultured with RTL VG1000 orTris-containing medium for 24 or 96 hr, collected and thoroughly washedwith RPMI, and then restimulated with 10 μg/ml hMOG-35-55 peptide in thepresence of DR81*1501-transfected L cells (A PC). After 48 hr, thesupernatants were transferred into IL-2-dependent CTLL cell cultures for24 hr and proliferation assessed by uptake of [³H]thymidine. As is shownin FIG. 6B, IL-2 secretion responses of the H2-1 hybrid cells tohMOG-35-55 peptide were inhibited 55% and 95% after respective 24 h or96 h co-cultures with RTL VG1000 compared to co-culture in medium alone.To control for non-specific inhibition, RTL VG 1000 was co-cultured inan identical manner with the A1 T cell hybrid specific for GP-M8P-69-89peptide (Gold et al., J. Exp. Med. 174:1467-76, 1991), and as is shownin FIG. 6B, no inhibition of the A 1 hybrid response to GP-M8P-69-89peptide was detected after a 24 h or 96 h incubation with RTL VG1000.These data demonstrate that the humanized Ag-specific T cell hybridsgenerated by from the DR2 Tg mice can be used as an unlimited cellsource to test in vitro biological functions of RTL preparations in anantigen-specific fashion.

To confirm that the inhibitory activity of RTL VG1000 preparationdetected by the H2-1 hybrid could be related to its inhibitory activityin vivo, the ability of RTL VG1000 to reverse EAE in DR2 Tg mice wasevaluated. Initially, hMOG-35-55 peptide was tested and shown to be bothimmunogenic and encephalitogenic in DRB1*1502 mice. The *1502 allelediffers from *1501 by only a G for V substitution at position 86 in theP1 binding pocket, but this substitution did not affect immunogenicityof known DR2-restricted peptides from neuroantigens or recall antigens.Following immunization with hMOG-35-55 in CFA, moderately severe EAE(score of about 2) was induced in a total of nine *1502-Tg mice. Afteronset of clinical signs, 3 mice were treated with 100 ug RTL VG1000given i.v. daily for 8 days, and 6 mice were treated under the sameconditions with Tris buffer (vehicle). As is shown in FIG. 6C, theseverity of EAE decreased to a score of 0.2±0.3 in mice treated withVG1000, whereas the severity of EAE increased to an average score of 4-5in vehicle-treated control mice. These in vivo test data demonstratedthe potent inhibitory effect of RTL VG 1000 on hMOG-35-55-induced EAE,and provided reference for the corresponding inhibitory effects of RTLVG1000 on the humanized hMOG-35-55 peptide-specific H2-1 hybrid.

Earlier studies showed that partial activation of a rat T cell hybrid byan Ag-specific RTL induced a TCRζ p23/p21 ratio shift, ZAP-70phosphorylation, calcium mobilization, NFAT activation, and transientIL-2 production (Wang et al., J. Immunol. 171:1934-40, 2003). Similarcharacterization will be conducted for the MOG-35-55-specific T cellhybrids of the invention to assess signal transduction events thatrelate to the inhibitory functions of RTLs. The finding of paired TCR αβgenes will facilitate analysis of the sequences of both VDJβ and VJα1.(Cα.) regions to determine the specific motifs associated withautoimmune encephalitogenic activities (Buenafe et al., J. Immunol.158:5472-83, 1997), and will allow a disclosure of the TCR sequences ofspecific genomic clones from our T cell hybrids. MOG-35-55 specifichybrids may also be valuable in screening and testing altered peptideligands that could induce cytokine shifts in human T cell clones(Bielekova et al., Nat. Med. 6:1167-75, 2000).

Additional characterization studies were conducted to elucidate thestarting T cell phenotype for optimal T cell hybrid construction. H2-1and BW5147 variant tumor cells 1×10⁶ per each were stained with 5 μl ofthe following rat monoclonal antibodies specific for mouseCD44-Cy-Chrome (clone IM7), CD62L-FITC (clone MEL-14), and CD45RB-PE(clone 16A), and an additional 0.5×10⁶ cells per each were stained with5 μl of hamster anti-mouse CD27-PE (clone LG.3A10) for 30 min at 40° C.Rat IgG2a-PE, Rat IgG2a-FITC, Rat IgG2b-Cy-Chrome, and Hamster IgG1-PEwere used for isotype control staining. After washing 2× with PBS, thecells were analyzed using Cell Quest™ software on a FACScan. Allmonoclonal antibodies were purchased from BD PharminGen (San Diego,Calif.).

According to these and other studies, the exemplary T hybrid H2-1 wasshown to have the following marker phenotype: CD45RB+, CD27+, CD62L+,CD44−

In additional work directed toward construction of human fusion partnercells for creating T hybrids, an HPRT deficient TCR-dependent Jurkat(J.RT3) mutant cell line was developed. This was initiated using alimited number of PBMC in primary peptide-stimulation followed byextended culture in rIL-2 to select T cell clones from a few HLA-DR2homozygous MS patients. A suitable human T cell tumor line J.RT3 waschosen as a starting fusion partner because of the CD3- andTCR-deficient phenotype of this line. However, the original J.RT3 linewas positive for HPRT, and therefore not useful as a fusion partner. TheHPRT-mutant of this line is generated at a low rate by irradiation andgrowth in 6-thioguanine-containing medium, which kills un-mutated J.RT3HPRT+cells.

For development of a J.RT3 derivative fusion partner, cell clonesspecific for MBP 85-99, PLP 139-151, MOG 35-55. All selected clones wereshown Ag-specific proliferation with a distinct TCR expression pattern.The J.RT3 line was irradiated for 1 min and cultured with increasedconcentrations of 6-thioguanine (up to 20 ug/ml) for 3 months to obtaina J.RT3 HPRT minus mutant cell line. The mutant J.RT3 line we createdhas kept the same growth rate as the original tumor line and maintainsits phenotype and no TCR cell surface expression same as the originalJ.RT3 line, but it can not survive in HAT-selecting medium as weexpected!

A total of 21 mature hybrids were generated. All hybrids expressed lowand varied amount of CD3 and TCR, though only in a few of them the CD3was significantly higher than that expressed by J.RT3 mutants. The datashow successful generation of human T cell hybrids from J.RT3 mutantfusion partners. Further development of the J.RT3 mutant fusion partnerswill lead to optimization of their use for generating human T cellhybrids having improved T cell phenotypes.

Deposit of Biological Materials

The exemplary T cell hybrid H2-1, as described herein above, has beendeposited with the American Type Culture Collection, (ATCC), Manasass,Va. and assigned Accession Number PTA-6082. This deposit will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. This deposit was made merely as a convenience for those ofskill in the art and is not an admission that a deposit is requiredunder 35 U.S.C. §112.

It will be understood by those skilled in the art that the foregoingdescription is intended to illustrate and not limit the scope of theinvention defined by the appended claims. Other aspects, advantages, andmodifications will be appreciated as embodied within the scope of theinvention.

1. A mammalian T cell hybrid comprising a fusion product of a mammalianT cell expressing a T cell receptor (TCR) and a mammalian fusion partnercell to yield a clonal T cell hybrid, wherein said T cell hybridexhibits an antigen (Ag)-specific, TCR-mediated proliferative responsefollowing contact of the T cell hybrid with a cognate Ag and anallogeneic Ag-presenting cell (APC).
 2. The T cell hybrid of claim 1,wherein the Ag-specific, TCR-mediated proliferative response of said Tcell hybrid following contact with the cognate Ag and APC can bedetectably inhibited or stimulated by contacting the T cell hybrid witha TCR antagonist or TCR agonist to yield a modified, Ag-specific,TCR-mediated proliferation response of the hybrid cell, wherein saidmodified proliferation response indicates a presence, quantity, and/oractivity level of the T cell antagonist or T cell agonist in contactwith the T cell hybrid.
 3. The T cell hybrid of claim 1, wherein theAg-specific, TCR-mediated proliferative response of said T cell hybridfollowing contact with the cognate Ag and APC can be detectablyinhibited or stimulated by contacting the T cell hybrid with arecombinant T cell receptor ligand (RTL) to yield a modified,Ag-specific, TCR-mediated proliferation response of the hybrid, whereinsaid modified proliferation response of the hybrid cell accurately andreproducibly indicates a presence, quantity, and/or activity level ofthe RTL in contact with the T cell hybrid.
 4. The T cell hybrid of claim4, wherein the T cell hybrid exhibits a Th1 cytokine expression profile.5. The T cell hybrid of claim 1, wherein the T cell expressing the TCRis a naïve T cell or a central memory T cell (Tcm).
 6. The T cell hybridof claim 1, wherein the T cell expressing the TCR is CD45+. 7.(canceled)
 8. (canceled)
 9. The T cell hybrid of claim 1, wherein T cellexpressing the TCR is CD62L+.
 10. The T cell hybrid of claim 1, whereinthe T cell expressing the TCR is CD27+.
 11. The T cell hybrid of claim1, wherein T cell expressing the TCR is CD49d−.
 12. The T cell hybrid ofclaim 1, wherein the T cell expressing the TCR is CCR7+.
 13. The T cellhybrid of claim 1, wherein the T cell expressing the TCR is LIGHT−.14-22. (canceled)
 23. The T cell hybrid of claim 22, wherein the T cellexpressing the TCR is transgenic for a HLA-DR, HLA-DP, or HLA-DQgenotype.
 24. The T cell hybrid of claim 23, wherein the T cellexpressing the TCR is transgenic for a mutant HLA-DR, HLA-DP, or HLA-DQisotype that contributes to a mammalian immune disorder. 25-27.(canceled)
 28. A mammalian T cell hybrid comprising a fusion product ofa mammalian T cell expressing a T cell receptor (TCR) and a mammalianfusion partner cell to yield a clonal T cell hybrid, wherein said T cellhybrid exhibits an antigen (Ag)-specific, TCR-mediated proliferativeresponse following contact of the T cell hybrid with a cognate Ag and anallogeneic Ag-presenting cell (APC), wherein said T cell expressing theTCR is transgenic for a mutant HLA-DR, HLA-DP, or HLA-DO isotype thatcontributes to a mammalian immune disorder, and wherein said mutantHLA-DR, HLA-DP, or HLA-DQ isotype is selected from a mutant HLA-DR1,HLA-DR2, HLA-DR3, HLA-DR4, HLA-DP2, HLA-DQ6, or HLA-DQ8 isotype.
 29. TheT cell hybrid of claim 28, wherein the mutant HLA-DR, HLA-DP, or HLA-DQisotype is selected from the group consisting of DRB1*0101; DRB1*0301,DRB1*0401, DRB1*0405, DRB1*1501, DRB4*0101, DRB5*0101, DPA1*0101,DPA1*0103, DPB1*0201, DPB1*0401, DQA1*0102, DQA1*0501, DQB1*0201,DQB1*0301, DQB1*0302, DQB1*0602, and DQB1*0604. 30-41. (canceled) 42.The T cell hybrid of claim 1, wherein said cognate Ag comprises animmunodominant T cell epitope associated with a mammalian autoimmunedisease, inflammatory disorder, allergic condition, cutaneous immunedisorder, transplant rejection condition, or graft versus host disease(GVHD).
 43. (canceled)
 44. The T cell hybrid of claim 42, wherein the Tcell epitope is associated with a mammalian autoimmune disease selectedfrom the group consisting of multiple sclerosis (MS), rheumatoidarthritis (RA), insulin-dependent diabetes mellitus (IDDM), chronicberyllium disease, autoimmune uveitis, sarcoidosis, systemic lupuserythromatosis, myasthenia gravis, and celiac disease.
 45. The T cellhybrid of claim 44, wherein the autoimmune disease is MS, and whereinthe immunodominant epitope is selected from the group consisting ofhuman (hu)-myelin oligodendrocyte protein (MOG)-1-22, huMOG-35-55,huMOG-huMOG-1-22, huMOG-34-54, huMOG-63-87, huMOG-64-96, huMOG-92-106,murine (mu)-MOG-1-30, muMOG-35-55, muMOG-8 1-110, muMOG-91-110, rat(rt)-MOG-1-20, rtMOG-35-55, rtMOG-74-90, guinea pig (Gp)-myelin basicprotein (MBP)-72-89, rt-MBP-72-89, hu-MBP-85-99, hu-MBP-86-99,hu-MBP-87-99, PLP-139-151, and hu type II collagen (CII)-259-273. 46-58.(canceled)
 59. The T cell hybrid of claim 1, wherein the mammalian Tcell expressing the TCR is isolated from a mammalian subject followingimmunization of the subject with a cognate Ag and prior todifferentiation of said T cell to express one or more markers expressedby an effector memory T cell (Tem) and not expressed at detectablelevels by naïve T lymphocytes and/or central memory T cells (Tcm).60-66. (canceled)
 67. The T cell hybrid of claim 1, wherein the T cellhybrid has the characteristics of hybrid cells deposited under ATCCaccession number: PTA-6082. 68-97. (canceled)